Nadh-dependent cytochrome b5 reductase as target for herbicides

ABSTRACT

The present invention relates to the use of a polypeptide with the biological activity of an NADH-dependent cytochrome b5 reductase (E.C. 1.6.2.2), which, when not present, brings about growth retardation symptoms and chlorotic leaves, and which is encoded by the nucleic acid sequence SEQ ID NO:1 or functional equivalents of the abovementioned nucleic acid sequence, as target for herbicides. Functional equivalents of SEQ ID NO:1 are provided in this context. Moreover, the present invention relates to the use of the polypeptide with the biological activity of an NADH-dependent cytochrome b5 reductase in a method for identifying herbicidally active compounds which inhibit NADH-dependent cytochrome b5 reductase. Moreover, the invention relates to the compounds identified by the method for use as herbicides.

The present invention relates to the use of a polypeptide with thebiological activity of an NADH-dependent cytochrome b5 reductase (E.C.1.6.2.2), which, when not present, brings about growth retardationsymptoms and chlorotic leaves, and which is encoded by the nucleic acidsequence SEQ ID NO:1 or functional equivalents of the abovementionednucleic acid sequence, as target for herbicides. Functional equivalentsSEQ ID NO:1 are provided in this context. Moreover, the presentinvention relates to the use of the polypeptide with the biologicalactivity of an NADH-dependent cytochrome b5 reductase in a method foridentifying herbicidally active compounds which inhibit NADH-dependentcytochrome b5 reductase. Moreover, the invention relates to thecompounds identified by the method for use as herbicides.

The basic principle of identifying herbicides by inhibiting a definedtarget is known (for example U.S. Pat. No. 5,187,071, WO 98/33925, WO00/77185). In general, there is a great demand for the detection ofenzymes which might constitute novel targets for herbicides. Reasonsherefore are that herbicidal active ingredients which act on knowntargets demonstrate the development of resistance problems, and theconstant endeavor to identify novel herbicidal active ingredients whichare distinguished by as broad as possible a range of action,ecofriendliness and toxicological compatibility and/or low applicationrates.

In practice, the detection of novel targets always entails greatdifficulties since the inhibition of an enzyme which is part of ametabolic pathway frequently has no further effect on the plant'sgrowth. The reason may be that the plant switches over to alternativemetabolic pathways whose existence is not known, or that the enzymewhich is being inhibited is not limiting for the metabolic pathway.Furthermore, plant genomes are distinguished by a high degree offunctional redundance. In the Arabidopsis thaliana genome, functionallyequivalent enzymes are more frequently found in gene families than isthe case with insects or mammals (Nature, 2000, 408(6814):796-815). Thishypothesis is confirmed experimentally by the fact that large geneknock-out programs by the insertion of T-DNA or transposons intoArabidopsis have, as yet, yielded fewer manifested phenotypes thanexpected (Curr. Op. Plant Biol. 4, 2001, pp. 111-117).

It is an object of the present invention to identify novel targets whichare essential for the growth of plants or whose inhibition leads toreduced plant growth, and to provide methods which are suitable foridentifying herbicidally active compounds.

We have found that this object is achieved by the use of a polypeptidewith the biological activity of an NADH-dependent cytochrome b5reductase encoded by a nucleic acid sequence consisting of

-   a) a nucleic acid sequence with the nucleic acid sequence shown in    SEQ ID NO:1; or-   b) a nucleic acid sequence which, on the basis of the degeneracy of    the genetic code, can be deduced from the amino acid sequence shown    in SEQ ID NO:2 by back translation; or-   c) a nucleic acid sequence which, on the basis of the degeneracy of    the genetic code, can be deduced from the amino acid sequence of a    functional equivalent of SEQ ID NO:2, which has at least 39%    identity with SEQ ID NO:2, by back translation; or-   d) a functional equivalent of the nucleic acid sequence SEQ ID NO:1    with at least 52% identity with SEQ ID NO:1    as target for herbicides.

Further terms used in the description are now defined at this point.

“Affinity tag”: This refers to a peptide or polypeptide whose codingnucleic acid sequence can be fused to the nucleic acid sequenceaccording to the invention either directly or by means of a linker,using customary cloning techniques. The affinity tag serves for theisolation, concentration and/or specific purification of the recombinanttarget protein by means of affinity chromatography from total cellextracts. The abovementioned linker can advantageously contain aprotease cleavage site (for example for Thrombin or Factor Xa), wherebythe affinity tag can be cleaved from the target protein when required.Examples of customary affinity tags are the “His tag”, for example fromQuiagen, Hilden, the “Strep tag”, the “Myc tag” (Invitrogen, Carlsberg),the tag from New England Biolabs which consists of a chitin-bindingdomain and an intein, the maltose-binding protein (pMal) from NewEngland Biolabs, and what is known as the CBD tag from Novagen. In thiscontext, the affinity tag can be attached to the 5′ or the 3′ end of thecoding nucleic acid sequence with the sequence encoding the targetprotein.

“Nucleic acid sequence according to the invention”: This term is definedfurther below.

“Expression cassette”: an expression cassette contains a nucleic acidsequence according to the invention linked operably to at least onegenetic control element, such as a promoter, and, advantageously, to afurther control element, such as a terminator. The nucleic acid sequenceof the expression cassette can be, for example, a genomic orcomplementary DNA sequence or an RNA sequence, and the semi- or fullysynthetic analogs thereof. These sequences can exist in linear orcircular form, extrachromosomally or integrated into the genome. Thenucleic acid sequences in question can be synthesized or obtainednaturally or contain a mixture of synthetic and natural DNA components,or else consist of various heterologous gene segments from variousorganisms.

Artificial nucleic acid sequences are also suitable in this context aslong as they make possible the expression, in a cell or an organism, ofa polypeptide with the biological activity of an NADH-dependentcytochrome b5 reductase, which polypeptide is encoded by a nucleic acidsequence according to the invention. For example, synthetic nucleotidesequences can be generated which have been optimized with regard to thecodon usage of the organisms to be transformed.

All of the abovementioned nucleotide sequences can be generated from thenucleotide units by chemical synthesis in the manner known per se, forexample by fragment condensation of individual overlapping complementarynucleotide units of the double helix. Oligonucleotides can besynthesized chemically for example in the manner known per se using thephosphoamidite method (Voet, Voet, 2^(nd) Edition, Wiley Press New York,pp. 896-897). When preparing an expression cassette, various DNAfragments can be manipulated in such a way that a nucleotide sequencewith the correct direction of reading and the correct reading frame isobtained. The nucleic acid fragments are linked with each other viageneral cloning techniques as are described, for example, in T.Maniatis, E. F. Fritsch and J. Sambrook, “Molecular Cloning: ALaboratory Manual”, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y. (1989), and in T. J. Silhavy, M. L. Berman and L. W. Enquist,Experiments with Gene Fusions, Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y. (1984) and in Ausubel, F. M. et al., “CurrentProtocols in Molecular Biology”, Greene Publishing Assoc. andWiley-Interscience (1994).

“Operable linkage”: an operable, or functional, linkage is understood asmeaning the sequential arrangement of regulatory sequences or geneticcontrol elements in such a way that each of the regulatory sequences, oreach of the genetic control elements, can fulfill its intended functionwhen the coding sequence is expressed.

“Functional equivalents” describe, in the present context, nucleic acidsequences which hybridize under standard conditions with the nucleicacid sequence SEQ ID NO:1 or parts of SEQ ID NO:1 and which are capableof bringing about the expression, in a cell or an organism, of apolypeptide with the biological activity of an NADH-dependent cytochromeb5 reductase.

To carry out the hybridization, it is advantageous to use shortoligonucleotides with a length of approximately 10-50 bp, preferably15-40 bp, for example of the conserved or other regions, which can bedetermined in the manner with which the skilled worker is familiar bycomparisons with other related genes. However, longer fragments of thenucleic acids according to the invention with a length of 100-500 bp, orthe complete sequences, may also be used for the hybridization.Depending on the nucleic acid/oligonucleotide used, or the length of thefragment or the complete sequence, or on the type of nucleic acid, i.e.DNA or RNA, that is being used for the hybridization, these standardconditions vary. Thus, for example, the melting temperatures for DNA:DNAhybrids are approximately 10° C. lower than those of DNA:RNA hybrids ofthe same length.

Standard hybridization conditions are to be understood as meaning,depending on the nucleic acid, for example temperatures of between 42and 58° C. in an aqueous buffer solution with a concentration of between0.1 and 5×SSC (1×SSC=0.15 M NaCl, 15 mM sodium citrate, pH 7.2) oradditionally in the presence of 50% formamide, such as, for example, 42°C. in 5×SSC, 50% formamide. The hybridization conditions for DNA:DNAhybrids are advantageously 0.1×SSC and temperatures of betweenapproximately 20° C. and 65° C., preferably between approximately 30° C.and 45° C. In the case of DNA:RNA hybrids, the hybridization conditionsare advantageously 0.1×SSC and temperatures of between approximately 30°C. and 65° C., preferably between approximately 45° C. and 55° C. Thesehybridization temperatures which have been stated are meltingtemperature values which have been calculated by way of example for anucleic acid with a length of approx. 100 nucleotides and a G+C contentof 50% in the absence of formamide. The experimental conditions for DNAhybridization are described in specialist textbooks of genetics such as,for example, in Sambrook et al., “Molecular Cloning”, Cold Spring HarborLaboratory, 1989, and can be calculated using formulae with which theskilled worker is familiar, for example as a function of the length ofthe nucleic acids, the type of the hybrids or the G+C content. Theskilled worker will find further information on hybridization in thefollowing text books: Ausubel et al. (eds), 1985, “Current Protocols inMolecular Biology”, John Wiley & Sons, New York; Hames and Higgins(eds), 1985, “Nucleic Acids Hybridization: A Practical Approach”, IRLPress at Oxford University Press, Oxford; Brown (ed), 1991, EssentialMolecular Biology: A Practical Approach, IRL Press at Oxford UniversityPress, Oxford.

A functional equivalent of the SEQ ID NO:1 is furthermore alsounderstood as meaning nucleic acid sequences which have up to a defineddegree of homology or identity with SEQ ID NO:1, and furthermore inparticular also natural or artificial mutations of the abovementionednucleic acid sequences which encode a polypeptide with the biologicalactivity of an NADH-dependent cytochrome b5 reductase.

For example, the present invention also encompasses, for example, thosenucleotide sequences which are obtained by modification of theabovementioned nucleic acid sequences. For example, such modificationscan be generated by techniques with which the skilled worker isfamiliar, such as “site directed mutagenesis”, “error prone PCR”,“DNA-shuffling” (Nature 370, 1994, pp. 389-391) or “staggered extensionprocess” (Nature Biotechnol. 16, 1998, pp. 258-261). The purpose of sucha modification can be, for example, the insertion of further cleavagesites for restriction enzymes, the removal of DNA in order to truncatethe sequence, the substitution of nucleotides to optimize the codons, orthe addition of further sequences. Proteins which are encoded viamodified nucleic acid sequences must retain the desired functionsdespite a deviating nucleic acid sequence.

The term “functional equivalent” can also relate to the amino acidsequence encoded by the nucleic acid sequence in question. In this case,the term “functional equivalent” describes a protein whose amino acidsequence has up to a defined percentage identity or homology with SEQ IDNO:2.

Functional equivalents thus comprise naturally occurring variants of theherein-described sequences and also artificial nucleic acid sequences,for example those which have been obtained by chemical synthesis andwhich are adapted to the codon usage, and also the amino acid sequencesderived from them.

“Genetic control sequence” describes sequences which have an effect onthe transcription and, if appropriate, translation of the nucleic acidsaccording to the invention in prokaryotic or eukaryotic organisms.Examples are promoters, terminators or what are known as “enhancer”sequences. In addition to these control sequences, or instead of thesesequences, the natural regulation of these sequences may still bepresent before the actual structural genes and may, if appropriate, havebeen genetically modified in such a way that the natural regulation hasbeen switched off and the expression of the target gene has beenmodified, that is to say increased or reduced. The choice of the controlsequence depends on the host organism or the starting organism. Geneticcontrol sequences furthermore also comprise the 5′-untranslated region,introns or the noncoding 3′ region of genes. Control sequences arefurthermore understood as meaning those which make possible homologousrecombination or insertion into the genome of a host organism, or whichpermit removal from the genome. Genetic control sequences also comprisefurther promoters, promoter elements or minimal promoters, and sequenceswhich have an effect on the chromatin structure (for example matrixattachment regions (MARs)), which can modify the expression-governingproperties. Thus, genetic control sequences may bring about, forexample, the fact that the tissue-specific expression is additionallydependent on certain stress factors. Such elements have been described,for example, for water stress, abscisic acid (Lam E and Chua N H, J BiolChem 1991; 266(26): 17131-17135), low temperature stress, drought stress(Plant Cell 1994, (6): 251-264) and high temperature stress (Molecular &General Genetics, 1989, 217(2-3): 246-53).

“Homology” between two nucleic acid sequences or polypeptide sequencesis defined by the identity of the nucleic acid sequence/polypeptidesequence over in each case the entire length of the sequence of theshorter sequence of the two, and this identity is calculated byalignment with the aid of the program algorithm GAP (Wisconsin PackageVersion 10.2 Genetics Computer Group (GCG), Madison, Wis., USA), settingthe following parameters for polypeptides Gap Weight: 8 Length Weight: 2Average Match: 2.912 Average Mismatch: −2.003

and the following parameters for nucleic acids: Gap Weight: 50 LengthWeight: 3 Average Match: 10.000 Average Mismatch: −0.000

In the following text, the term “identity” is also used synonymouslywith the term “homologous” or “homology”.

“Mutations” of nucleic or amino acid sequences encompass substitutions,additions, deletions, inversions or insertions of one or more nucleotideresidues, which may also bring about changes in the corresponding aminoacid sequence of the target protein by substitution, insertion ordeletion of one or more amino acids, but where the functional propertiesof the target protein in total are essentially retained.

“Natural genetic environment” refers to the natural chromosomal locus inthe organism of origin. In the case of a genomic library, the naturalgenetic environment of the nucleic acid sequence is preferably retainedat least in part. The environment flanks the nucleic acid sequence atleast at 5′ or 3′ and has a sequence length of at least 50 bp,preferably at least 100 bp, especially preferably at least 500 bp, veryespecially preferably at least 1000 bp, most preferably at least 5000bp.

“Plants” for the purposes of the invention are plant cells, planttissues, plant organs, or intact plants, such as seeds, tubers, flowers,pollen, fruits, seedlings, roots, leaves, stems or other plant parts.Moreover, the term plants is understood as meaning propagation materialsuch as seeds, fruits, seedlings, slips, tubers, cuttings or rootstocks.

“Reaction time” refers to the time required for carrying out an activityassay until a significant finding regarding an activity is obtained; itdepends both on the specific activity of the protein employed in theassay and on the method used and the sensitivity of the apparatus used.The skilled worker is familiar with the determination of the reactiontimes. In the case of methods for identifying herbicidally activecompounds which are based on photometry, the reaction times are, forexample, between >0 and 120 minutes.

“Recombinant DNA” describes a combination of DNA sequences which can begenerated by recombinant DNA technology.

“Recombinant DNA technology”: generally known techniques for fusing DNAsequences (for example described in Sambrook et al., 1989, Cold SpringHabour, N.Y., Cold Spring Habour Laboratory Press).

“Replication origins” ensure the replication of the expression cassettesor vectors according to the invention in microorganisms and yeasts, forexample the pBR322 ori or the P15A ori in E. coli (Sambrook et al.:“Molecular Cloning. A Laboratory Manual”, 2nd ed. Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989) and the ARS1 ori inyeast (Nucleic Acids Research, 2000, 28(10): 2060-2068).

“Reporter genes” encode readily quantifiable proteins. Thetransformation efficacy or the transformation site or timing can beassessed by means of these genes via a growth assay, fluorescence assay,chemoluminescence assay, bioluminescence assay or resistance assay orvia a photometric measurement (intrinsic color) or enzyme activity. Veryespecially preferred in this context are reporter proteins (Schenborn E,Groskreutz D. Mol Biotechnol. 1999; 13(1):29-44) such as the “greenfluorescence protein” (GFP) (Gerdes H H and Kaether C, FEBS Lett. 1996;389(1):44-47; Chui W L et al., Curr Biol 1996, 6:325-330; Leffel S M etal., Biotechniques. 23(5):912-8, 1997), chloramphenicolacetyltransferase, a luciferase (Giacomin, Plant Sci 1996, 116:59-72;Scikantha, J Bact 1996, 178:121; Millar et al., Plant Mol Biol Rep 199210:324-414), and luciferase genes, in general β-galactosidase orβ-glucuronidase (Jefferson et al., EMBO J. 1987, 6, 3901-3907) or theUra3 gene.

“Selection markers” confer a resistance to antibiotics or other toxiccompounds: examples which may be mentioned in this context are theneomycin phosphotransferase gene, which confers resistance to theaminoglycoside antibiotics neomycin (G 418), kanamycin, paromycin(Deshayes A et al., EMBO J. 4 (1985) 2731-2737), the sul gene encoding amutated dihydropteroate synthase (Guerineau F et al., Plant Mol Biol.1990; 15(1):127-136), the hygromycin B phosphotransferase gene (Gen BankAccession NO: K 01193) and the shble resistance gene, which confersresistance to the bleomycin antibiotics such as, for example, zeocin.Further examples of selection marker genes are genes which conferresistance to 2-deoxyglucose-6-phosphate (WO 98/45456) orphosphinothricin and the like, or those which confer a resistance toantimetabolites, for example the dhfr gene (Reiss, Plant Physiol. (LifeSci. Adv.) 13 (1994) 142-149). Examples of other genes which aresuitable are trpB or hisD (Hartman S C and Mulligan R C, Proc Natl AcadSci USA. 85 (1988) 8047-8051). Another suitable gene is themannose-phosphate isomerase gene (WO 94/20627), the ODC (ornithindecarboxylase) gene (McConlogue, 1987 in: Current Communications inMolecular Biology, Cold Spring Harbor Laboratory, Hrsg.) or theAspergillus terreus deaminase (Tamura K et al., Biosci BiotechnolBiochem. 59 (1995) 2336-2338).

“Transformation” describes a process for introducing heterologous DNAinto a prokaryotic or eukaryotic cell. The term transformed celldescribes not only the product of the transformation process per se, butalso all of the transgenic progeny of the transgenic organism generatedby the transformation.

“Target/target protein”: a polypeptide encoded by the nucleic acidsequence according to the invention, which may take the form of anenzyme in the traditional sense or, for example, of a structuralprotein, development protein, regulatory proteins such as transcriptionfactors, kinases, phosphatases, receptors, channel subunits, transportproteins, regulatory subunits which confer substrate or activityregulation to an enzyme complex. All of the targets or sites of actionshare the characteristic that their functional presence is essential forthe survival or the normal development and growth.

“Transgenic”: referring to a nucleic acid sequence, an expressioncassette or a vector comprising a nucleic acid sequence according to theinvention or an organism transformed with the abovementioned nucleicacid sequence, expression cassette or vector, the term transgenicdescribes all those constructs which have been generated by recombinantmethods in which either the nucleic acid sequence of the target proteinor a genetic control sequence linked operably to the nucleic acidsequence of the target protein or a combination of the abovementionedpossibilities are not in their natural genetic environment or have beenmodified by recombinant methods. In this context, the modification canbe achieved, for example, by mutating one or more nucleotide residues ofthe nucleic acid sequence in question.

The nucleic acid sequence SEQ ID NO:1 encodes a specificallyNADH-dependent cytochrome b5 reductase (E.C. 1.6.2.2).

The electron transfer system located at the membrane of the endoplasmicreticulum, which is characteristic of higher eukaryotes, consists of anNADH-dependent cytochrome b5 reductase and cytochrome b5 (Cytb5). TheNADH-dependent cytochrome b5 reductase, in which FAD is the prostheticgroup, transfers electrons from NADH to Cytb5, a heme-containingprotein. Thus, polypeptide with the biological activity of anNADH-dependent cytochrome b5 reductase refers to an enzyme which iscapable, with an FAD as prosthetic group, of transferring electrons fromNADH to Cytb5, a heme-containing protein. The enzymatic activity of anenzyme with the biological activity of an NADH-dependent cytochrome b5reductase can be determined by means of suitable activity assays as aredescribed by way of example further below (see also Example 5, interalia).

In plants, Cytb5 was described as component of the electron transport inthe modification of fatty acids (Kearns et al., 1991; SpektrumAkademischer Verlag Heidelberg, Berlin, Oxford, pp. 751). It is assumedthat the electrons are subsequently transferred from cytochrome b5 todesaturases or P450 monooxygenases (Fukuchi-Mizutani, Plant Physiology,119; 353-361; 1999). Plant NADH-dependent cytochrome b5 reductases arefound in virtually all cell types, in particular in immature seeds(Fukuchi-Mizutani, Plant Physiology, 119; 353-361; 1999).

The NADH-dependent cytochrome b5 reductase compound was first isolatedand characterized from human erythrocytes (Yubisui T, Takeshita M., JBiol Chem., 1980;255(6):2454-1456) and since then from a large number ofother organisms. Nucleic acid sequences of plant NADH-dependentcytochrome b5 reductases are known from, for example, Arabidopsis (GenBank Acc. No. AB007799; Mizutani and Fukuchi-Mizutani, Plant Physiol.119, 353-361; 1999) and ESTs of plant NADH-dependent cytochrome b5reductases from Medicago truncatula (Gen Bank Acc. No. AA660929; Covitz,P. A. et al. Plant Physiol. 117 (4), 1325-1332 (1998) Identity with SEQID NO:1=68.763%, Identity with SEQ ID NO:2=46.897%), Oryza sativa (GenBank Acc. No. BE039960; Identity with SEQ ID NO:1=69.457%, Identity withSEQ ID NO:2=75.912%), Solanum tuberosum (Gen Bank Acc. No. BE340917,Identity with SEQ ID NO:1=75.564%, Identity with SEQ ID NO:2=81.675%)and Beta vulgaris (Gen Bank Acc. No. BI096337; Identity with SEQ IDNO:1=52.727%; Identity with SEQ ID NO:2=39.552%) and squash (Cucurbitamaxima; Gen Bank Acc. No. AF274589; Identity with SEQ ID NO:1=56.703%,Identity with SEQ ID NO:2=43.621%).

The NADH-dependent cytochrome b5 reductase from human erthyrocytes canbe inhibited by inositol hexaphosphate concentrations in the millimolarrange (Palmieri et al., Archives of Biochemistry and Biophysics, 1990,280(1), 224-228). At a concentration of 0.5 mM, thenoyltrifluoroacetoneshows 50% inhibition of NADH-dependent cytochrome b5 reductase from ratliver microsomes (Golf et al., Biol. Chem. Hoppe-Seyler 1985, 366,647-653). Other substances which are capable of inhibitingNADH-dependent cytochrome b5 reductase from various organisms areamytal, mepacrin, dicoumarol (Golf et al.; 1985, Biol. Chem.Hoppe-Seyler, 366, pp. 647-653), or N-ethylmaleimide and atebrin (Tamuraet al.; 1983, J. Biochem. 94, pp. 1547-1555). However, inhibitors forplant NADH-dependent cytochrome b5 reductases have not been described todate.

Surprisingly, it has been found within the scope of the presentinvention that plants in which the activity of NADH-dependent cytochromeb5 reductase was reduced in a targeted fashion displayed phenotypeswhich are comparable with phenotypes generated by the application ofherbicide. Among the symptoms observed were growth retardation andnecrotic, stressed leaves and, in some cases, the death of entireplants, or of plant parts. The pods of these plants were either empty orcontained shriveled seeds, none of which was capable of germination.

The present invention relates to the use of a polypeptide with thebiological activity of an NADH-dependent cytochrome b5 reductase encodedby a nucleic acid sequence consisting of

-   a) a nucleic acid sequence with the nucleic acid sequence shown in    SEQ ID NO:1; or;-   b) a nucleic acid sequence which, on the basis of the degeneracy of    the genetic code, can be deduced from the amino acid sequence shown    in SEQ ID NO:2 by back translation; or-   c) a nucleic acid sequence which, on the basis of the degeneracy of    the genetic code, can be deduced from the amino acid sequence of a    functional equivalent of SEQ ID NO:2, which has at least 39%    identity with SEQ ID NO:2, by back translation; or-   d) a functional equivalent of the nucleic acid sequence SEQ ID NO:1    with at least 52% identity with SEQ ID NO:1;    as target for herbicides. The functional equivalents of c) are    distinguished by an essentially identical functionality, i.e. they    have the physiological function of an NADH-dependent cytochrome b5    reductase.

The functional equivalents according to the invention of SEQ ID NO:1have at least 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, by reference atleast 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69% and 70%,preferably at least 71%, 72%, 73%, 74%, 75%, 76%, especially preferablyat least 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, very especially preferably at least 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% homology with SEQ ID NO:1.

The functional equivalents according to the invention of SEQ ID NO:2have at least 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%,50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, by preference at least60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69% and 70%, preferably atleast 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, especially preferably at least 87%, 88%, 89%, 90%, 91%,92%, 93%, very especially preferably at least 94%, 95%, 96%, 97%, 98%,99% homology with SEQ ID NO:2.

Examples of functional equivalents are the plant nucleic acid sequenceswhich encode NADH-dependent cytochrome b5 reductase and which havealready been mentioned further above or amino acid sequences of anNADH-dependent cytochrome b5 reductase from Medicago truncatula (GenBank Acc. No. AA660929; Covitz, P. A. et al. Plant Physiol. 117 (4),1325-1332 (1998)), Oryza sativa (Gen Bank Acc. No. BE039960), Solanumtuberosum (Gen Bank Acc. No. BE340917), Beta vulgaris (Gen Bank Acc. No.BI096337) and squash (Cucurbita maxima; Gen Bank Acc. No. AF274589).

Moreover, the present invention claims functional equivalents of theabovementioned nucleic acid sequences which encode a polypeptide withthe biological activity of an NADH-dependent cytochrome b5 reductasecomprising a part-region encompassing:

-   a) a nucleic acid sequence with the nucleic acid sequence shown in    SEQ ID NO:3; or-   b) a nucleic acid sequence which, on the basis of the degeneracy of    the genetic code, can be deduced from the amino acid sequence shown    in SEQ ID NO:4 by back translation; or-   c) functional equivalents of the nucleic acid sequence SEQ ID NO:3    with at least 77% identity with SEQ ID NO:3; or-   d) a nucleic acid sequence which, on the basis of the degeneracy of    the genetic code, can be deduced from the amino acid sequence of a    functional equivalent of SEQ ID NO:4, which has at least 87%    identity with SEQ ID NO:4; by back translation.

The polypeptides encoded by the abovementioned nucleic acid sequencesare likewise claimed. The functional equivalents are distinguished by anessentially identical functionality, i.e. they have the physiologicalfunction of an NADH-dependent cytochrome b5 reductase.

The term “encompassing” or “to encompass” in the context of nucleic acidsequences refers to the fact that the nucleic acid sequence according tothe invention may contain additional nucleic acid sequences at the 3′ or5′ end, the length of the additional nucleic acid sequences notexceeding 75 bp of the 5′ end and 50 bp of the 3′ end, preferably 50 bpat the 5′ end and 10 bp at the 3′ end, of the nucleic acid sequencesaccording to the invention.

The functional equivalents according to the invention of SEQ ID NO:3have at least 77%, 78%, 79%, 80%, preferably at least 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, especially preferably at least 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% homology with SEQ ID NO:3.

The functional equivalents according to the invention of SEQ ID NO:4have at least 87%, by preference at least 88%, 89%, preferably at least90%, 91%, 92%, 93%, especially preferably at least 94%, 95%, 96%, veryespecially preferably at least 97%, 98%, 99% homology with SEQ ID NO:4.

Nucleic acid sequences encoding a polypeptide with the biologicalactivity of an NADH-dependent cytochrome b5 reductase consisting of

-   a) a nucleic acid sequence with the nucleic acid sequence shown in    SEQ ID NO:1; or-   b) a nucleic acid sequence which, on the basis of the degeneracy of    the genetic code, can be deduced from the amino acid sequence shown    in SEQ ID NO:2 by back translation; or-   c) a nucleic acid sequence which, on the basis of the degeneracy of    the genetic code, can be deduced from the amino acid sequence of a    functional equivalent of SEQ ID NO:2, which has at least 39%    identity with SEQ ID NO:2, by back translation; or-   d) a functional equivalent of the nucleic acid sequence SEQ ID NO:1    with at least 52% identity with SEQ ID NO:1; or    nucleic acid sequences encoding a polypeptide with the biological    activity of an NADH-dependent cytochrome b5 reductase comprising a    part-region encompassing:-   a) a nucleic acid sequence with the nucleic acid sequence shown in    SEQ ID NO:3; or-   b) a nucleic acid sequence which, on the basis of the degeneracy of    the genetic code, can be deduced from the amino acid sequence shown    in SEQ ID NO:4 by back translation; or-   c) functional equivalents of the nucleic acid sequence SEQ ID NO:3    with at least 86% identity with SEQ ID NO:3; or-   d) a nucleic acid sequence which, on the basis of the degeneracy of    the genetic code, can be deduced from the amino acid sequence of a    functional equivalent of SEQ ID NO:4, which has at least 87%    identity with SEQ ID NO:4, by back translation;    are hereinbelow referred to as “nucleic acid sequences according to    the invention. The polypeptides encoded by a nucleic acid sequence    according to the invention with the biological activity of an    NADH-dependent cytochrome b5 reductase are hereinbelow referred to    as “NCRs” for the sake of simplicity.

NCRs, in reduced quantity, cause growth retardation symptoms andnecrotic leaves in plants. A reduction in the polypeptide means that theamount of the polypeptide is reduced by recombinant methods. A plantwhich has been modified thus is compared with a plant which has not beengenetically modified with regard to this polypeptide, but which isotherwise identical with the genotype of the genetically manipulatedplant under identical growth conditions.

The gene products of the nucleic acids according to the inventionconstitute novel targets for herbicides which allow novel herbicides forcontrolling undesired plants to be provided.

Undesired plants are understood as meaning, in the broadest sense, allthose plants which grow at locations where they are undesired, forexample:

Dicotyledonous weeds of the genera: Sinapis, Lepidium, Galium,Stellaria, Matricaria, Anthemis, Galinsoga, Chenopodium, Urtica,Senecio, Amaranthus, Portulaca, Xanthium, Convolvulus, Ipomoea,Polygonum, Sesbania, Ambrosia, Cirsium, Carduus, Sonchus, Solanum,Rorippa, Rotala, Lindernia, Lamium, Veronica, Abutilon, Emex, Datura,Viola, Galeopsis, Papaver, Centaurea, Trifolium, Ranunculus, Taraxacum.

Monocotyledonous weeds from the genera: Echinochloa, Setaria, Panicum,Digitaria, Phleum, Poa, Festuca, Eleusine, Brachiaria, Lolium, Bromus,Avena, Cyperus, Sorghum, Agropyron, Cynodon, Monochoria, Fimbristyslis,Sagittaria, Eleocharis, Scirpus, Paspalum, Ischaemum, Sphenoclea,Dactyloctenium, Agrostis, Alopecurus, Apera.

SEQ ID NO:1 or SEQ ID NO:3 or parts of the abovementioned nucleic acidsequences can be used for the preparation of hybridization probes, bymeans of which for example the corresponding full-length genes and/orfunctional equivalents of SEQ ID NO:1 or SEQ ID NO:3 can be isolated.The preparation of these probes and the experimental procedure is known.For example, this can be effected via the tailor-made preparation ofradioactive or nonradioactive probes by means of PCR and the use ofsuitably labeled oligonucleotides, followed by hybridizationexperiments. The technologies required for this purpose are given, forexample, in T. Maniatis, E. F. Fritsch and J. Sambrook, “MolecularCloning: A Laboratory Manual”, Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y. (1989). The probes in question can furthermore bemodified by standard technologies (Lit. SDM or random mutagenesis) sothat they can be employed for further purposes, for example as probewhich hybridizes specifically with mRNA and the corresponding codingsequences, in order to analyze the corresponding sequences in otherorganisms.

Moreover, the abovementioned probes can be used for the detection andisolation of functional equivalents of SEQ ID NO:1 or SEQ ID NO:3 fromother plant species on the basis of sequence identities. In thiscontext, part or all of the sequence of corresponding SEQ ID NO:1 or SEQID NO:3 is used as probe for screening in a genomic library or cDNAlibrary of the plant species in question or in a computer search forsequences of functional equivalents in electronic databases.

Preferred plant species are the undesired plants which have already beenmentioned at the outset.

The invention furthermore relates to expression cassettes comprising

-   a) genetic control sequences in functional linkage with a nucleic    acid sequence encompassing a part-region comprising a nucleic acid    sequence with the nucleic acid sequence shown in SEQ ID NO:3; or a    nucleic acid sequence which, on the basis of the degeneracy of the    genetic code, can be deduced from the amino acid sequence shown in    SEQ ID NO:4 by back translation; or functional equivalents of the    nucleic acid sequence SEQ ID NO:3 with at least 86% identity with    SEQ ID NO:3; a nucleic acid sequence which, on the basis of the    degeneracy of the genetic code, can be deduced from the amino acid    sequence of the functional equivalent of SEQ ID NO:4, which has at    least 87% identity with SEQ ID NO:4, by back translation;-   b) additional functional elements; or-   c) a combination of a) and b);    and to the use of expression cassettes comprising-   a) genetic control sequences in operable linkage with a nucleic acid    sequence according to the invention;-   b) additional functional elements; or-   c) a combination of a) and b);    for expressing an NCR which can be used in “in vitro” assay systems.    Both embodiments of the above-described expression cassettes are    hereinbelow referred to as expression cassettes according to the    invention.

In a preferred embodiment, an expression cassette according to theinvention comprises a promoter at the 5′ end of the coding sequence and,at the 3′ end, a transcription termination signal and, if appropriate,further genetic control sequences which are linked operably with theinterposed nucleic acid sequence according to the invention.

The expression cassettes according to the invention are also understoodas meaning analogs which can be brought about, for example, by acombination of individual nucleic acid sequences on a polynucleotide(multiple constructs, on a plurality of polynucleotides in a cell(cotransformation) or by sequential transformation.

Advantageous genetic control sequences under item a) for the expressioncassettes according to the invention or for vectors comprisingexpression cassettes according to the invention are, for example,promoters such as cos, tac, trp, tet, lpp, lac, laciq, T7, T5, T3, gal,trc, ara, SP6, λ-PR or the λ-PL promoter, all of which can be used forexpressing NCR in Gram-negative bacterial strains.

Examples of further advantageous genetic control sequences are present,for example, in the promoters amy and SPO2, both of which can be usedfor expressing NCR in Gram-positive bacterial strains, and in the yeastor fungal promoters AUG1, GPD-1, PX6, TEF, CUP1, PGK, GAP1, TPI, PHO5,AOX1, GAL10/CYC1, CYC1, OliC, ADH, TDH, Kex2, MFa or NMT or combinationsof the abovementioned promoters (Degryse et al., Yeast 1995 Jun. 15;11(7):629-40; Romanos et al. Yeast 1992 June;8(6):423-88; Benito et al.Eur. J. Plant Pathol. 104, 207-220 (1998); Cregg et al. Biotechnology(N.Y.) 1993 August;11(8):905-10; Luo X., Gene 1995 Sep.22;163(1):127-31; Nacken et al., Gene 1996 Oct. 10;175(1-2): 253-60;Turgeon et al., Mol Cell Biol 1987 September;7(9):3297-305) or thetranscription terminators NMT, Gcy1, TrpC, AOX1, nos, PGK or CYC1(Degryse et al., Yeast 1995 Jun. 15; 11(7):629-40; Brunelli et al. Yeast1993 Dec. 9(12): 1309-18; Frisch et al., Plant Mol. Biol. 27 (2),405-409 (1995); Scorer et al., Biotechnology (N.Y.) 12 (2), 181-184(1994), Genbank Acc. number Z46232; Zhao et al. Genbank Acc number:AF049064; Punt et al., (1987) Gene 56 (1), 117-124), all of which can beused for expressing NCR in yeast strains.

Examples of genetic control sequences which are suitable for expressionin insect cells are the polyhedrin promoter and the p10 promoter(Luckow, V. A. and Summers, M. D. (1988) Bio/Techn. 6, 47-55).

Advantageous genetic control sequences for expressing NCR in cellculture are, in addition to polyadenylation sequences such as, forexample, from simian virus 40, eukaryotic promoters of viral origin suchas, for example, promoters of the polyoma virus, adenovirus 2,cytomegalovirus or simian virus 40.

Further advantageous genetic control sequences for expressing NCR inplants are present in the plant promoters CaMV/35S [Franck et al., Cell21(1980) 285-294], PRP1 [Ward et al., Plant. Mol. Biol. 22 (1993)], SSU,OCS, LEB4, USP, STLS1, B33, NOS; FBPaseP (WO 98/18940) or in theubiquitin or phaseolin promoter; a promoter which is preferably usedbeing, in particular, a plant promoter or a promoter derived from aplant virus. Especially preferred are promoters of viral origin, such asthe promoter of the cauliflower mosaic virus 35S transcript (Franck etal., Cell 21 (1980), 285-294; Odell et al., Nature 313 (1985), 810-812).Further preferred constitutive promoters are, for example, theAgrobacterium nopaline synthase promoter, the TR dual promoter, theAgrobacterium OCS (octopine synthase) promoter, the ubiquitin promoter(Holtorf S et al., Plant Mol Biol 1995, 29:637-649), the promoters ofthe vacuolar ATPase subunits, or the promoter of a proline-rich proteinfrom wheat (WO 91/13991).

The expression cassettes may also comprise, as genetic control sequence,a chemically inducible promoter, by means of which the expression of theexogenous gene in the plant can be controlled at a specific point intime. Such promoters, such as, for example, the PRP1 promoter (Ward etal., Plant. Mol. Biol. 22 (1993), 361-366), a salicylic-acid-induciblepromoter (WO 95/19443), a benzenesulfonamide-inducible promoter(EP-A-0388186), a tetracyclin-inducible promoter (Gatz et al., (1992)Plant J. 2, 397404), an abscisic-acid-inducible promoter (EP-A 335528)or an ethanol- or cyclohexanone-inducible promoter (WO 93/21334) mayalso be used.

Furthermore, suitable promoters are those which confer tissue- ororgan-specific expression in, for example, anthers, ovaries,inflorescences and floral organs, leaves, stomata, trichomes, stems,vascular tissues, roots and seeds. Others which are suitable in additionto the abovementioned constitutive promoters are, in particular, thosepromoters which ensure leaf-specific expression. Promoters which must bementioned are the potato cytosolic FBPase promoter (WO 97/05900), therubisco (ribulose-1,5-bisphosphate carboxylase) SSU (small subunit)promoter or the ST-LSI promoter from potato (Stockhaus et al., EMBO J. 8(1989), 2445-245). Promoters which are furthermore preferred are thosewhich control expression in seeds and plant embryos. Examples ofseed-specific promoters are the phaseolin promoter (U.S. Pat. No.5,504,200, Bustos M M et al., Plant Cell. 1989;1(9):839-53), thepromoter of the 2S albumin gene (Joseffson L G et al., J Biol Chem 1987,262:12196-12201), the legumin promoter (Shirsat A et al., Mol Gen Genet.1989;215(2):326-331), the USP (unknown seed protein) promoter (BaumleinH et al., Molecular & General Genetics 1991, 225(3):459-67), the napingene promoter (Stalberg K, et al., L. Planta 1996, 199:515-519), thesucrose binding protein promoter (WO 00/26388) or the LeB4 promoter(Baumlein H et al., Mol Gen Genet 1991, 225: 121-128; Fiedler, U. etal., Biotechnology (NY) (1995), 13 (10) 1090).

Further promoters which are suitable as genetic control sequences are,for example, specific promoters for tubers, storage roots or roots, suchas, for example, the class I patatin promoter (B33), the potatocathepsin D inhibitor promoter, the starch synthase (GBSS1) promoter orthe sporamin promoter, fruit-specific promoters such as, for example,the fruit-specific promoter from tomato (EP-A 409625),fruit-maturation-specific promoters such as, for example, thefruit-maturation-specific promoter from tomato (WO 94/21794),inflorescence-specific promoters such as, for example, the phytoenesynthase promoter (WO 92/16635) or the promoter of the P-rr gene (WO98/22593), or specific plastid or chromoplast promoters such as, forexample, the RNA polymerase promoter (WO 97/06250), or else the Glycinemax phosphoribosyl-pyrophosphate amidotransferase promoter (see alsoGenbank Accession No. U87999), or another node-specific promoter asdescribed in EP-A 249676 may be used advantageously.

Additional functional elements b) are understood as meaning by way ofexample but not by limitation reporter genes, replication origins,selection markers and what are known as affinity tags, in fusion withNCR directly or by means of a linker optionally comprising a proteasecleavage site. Further suitable additional functional elements aresequences which ensure that the product is targeted into the apoplasts,into plastids, the vacuoles, the mitochondrion, the peroxisome, theendoplasmic reticulum (ER) or, owing to the absence of such operativesequences, remains in the compartment where it is formed, the cytosol(Kermode, Crit. Rev. Plant Sci. 15, 4 (1996), 285-423).

Also in accordance with the invention are vectors comprising at leastone copy of the nucleic acid sequences according to the invention and/orthe expression cassettes according to the invention.

In addition to plasmids, vectors are furthermore also understood asmeaning all of the other known vectors with which the skilled worker isfamiliar, such as, for example, phages, viruses such as SV40, CMV,baculovirus, adenovirus, transposons, IS elements, phasmids, phagemids,cosmids or linear or circular DNA. These vectors can replicateautonomously in the host organism or replicate chromosomally;chromosomal replication is preferred.

In a further embodiment of the vector, the nucleic acid constructaccording to the invention can advantageously also be introduced intothe organisms in the form of a linear DNA and integrated into the genomeof the host organism via heterologous or homologous recombination. Thislinear DNA may consist of a linearized plasmid or only of the nucleicacid construct as vector, or the nucleic acid sequences used.

Further prokaryotic or eukaryotic expression systems are mentioned inChapters 16 and 17 in Sambrook et al., “Molecular Cloning: A LaboratoryManual.” 2nd ed., Cold Spring Harbor Laboratory, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989. Further advantageousvectors are described in Hellens et al. (Trends in plant science, 5,2000).

The expression cassette according to the invention and vectors derivedtherefrom can be used for transforming bacteria, cyanobacteria, yeasts,filamentous fungi and algae and eukaryotic nonhuman cells (for exampleinsect cells) with the aim of producing NCR recombinantly, thegeneration of a suitable expression cassette depending on the organismin which the gene is to be expressed.

In a further advantageous embodiment, the nucleic acid sequences used inthe method according to the invention may also be introduced into anorganism by themselves.

If, in addition to the nucleic acid sequences, further genes are to beintroduced into the organism, they can all be introduced into theorganism together in a single vector, or each individual gene can beintroduced into the organism in in each case one vector, it beingpossible to introduce the different vectors simultaneously or insuccession.

In this context, the introduction, into the organisms in question(transformation), of the nucleic acid(s) according to the invention, ofthe expression cassette or of the vector can be effected in principle byall methods with which the skilled worker is familiar.

In the case of microorganisms, the skilled worker will find suitablemethods in the textbooks by Sambrook, J. et al. (1989) “Molecularcloning: A laboratory manual”, Cold Spring Harbor Laboratory Press, byF. M. Ausubel et al. (1994) “Current protocols in molecular biology”,John Wiley and Sons, by D. M. Glover et al., DNA Cloning Vol. 1, (1995),IRL Press (ISBN 019-963476-9), by Kaiser et al. (1994) Methods in YeastGenetics, Cold Spring Harbor Laboratory Press or Guthrie et al. “Guideto Yeast Genetics and Molecular Biology”, Methods in Enzymology, 1994,Academic Press.

In the case of dicots, the methods which have been described for thetransformation and regeneration of plants from plant tissues or plantcells can be exploited for transient or stable transformation. Suitablemethods are the biolistic method or the transformation of protoplasts(cf., for example, Willmitzer, L., 1993 Transgenic plants. In:Biotechnology, A Multi-Volume Comprehensive Treatise (H. J. Rehm, G.Reed, A. Pühler, P. Stadler, eds.), Vol. 2, 627-659, VCH Weinheim-NewYork-Basel-Cambridge), electroporation, the incubation of dry embryos inDNA-containing solution, microinjection and the agrobacterium-mediatedgene transfer. The abovementioned methods are described, for example, inB. Jenes et al., Techniques for Gene Transfer, in: Transgenic Plants,Vol. 1, Engineering and Utilization, edited by S. D. Kung and R. Wu,Academic Press (1993) 128-143 and in Potrykus, Annu. Rev. Plant Physiol.Plant Molec. Biol. 42 (1991) 205-225).

The transformation by means of agrobacteria, and the vectors to be usedfor the transformation, are known to the skilled worker and describedextensively in the literature (Bevan et al., Nucl. Acids Res. 12 (1984)8711. The intermediary vectors can be integrated into the agrobacterialTi or Ri plasmid by means of homologous recombination owing to sequenceswhich are homologous to sequences in the T-DNA. This plasmidadditionally contains the vir region, which is required for the transferof the T-DNA. Intermediary vectors are not capable of replication inagrobacteria. The intermediary vector can be transferred toAgrobacterium tumefaciens by means of a helper plasmid (conjugation).Binary vectors are capable of replication both in E. coli and inagrobacteria. They contain a selection marker gene and a linker orpolylinker which are framed by the right and left T-DNA border region.They can be transformed directly into the agrobacteria (Holsters et al.Mol. Gen. Genet. 163 (1978), 181-187), EP A 0 120 516; Hoekema, In: TheBinary Plant Vector System Offsetdrukkerij Kanters B. V., Alblasserdam(1985), Chapter V; Fraley et al., Crit. Rev. Plant. Sci., 4: 1-46 and Anet al. EMBO J. 4 (1985), 277-287).

The transformation of monocots by means of vectors based onAgrobacterium has also been described (Chan et al., Plant Mol. Biol.22(1993), 491-506; Hiei et al., Plant J. 6 (1994) 271-282; Deng et al.;Science in China 33 (1990), 28-34; wilmink et al., Plant Cell Reports11, (1992) 76-80; May et al.; Biotechnology 13 (1995) 486-492; Connerand Domisse; Int. J. Plant Sci. 153 (1992) 550-555; Ritchie et al.;Transgenic Res. (1993) 252-265). Alternative systems for thetransformation of monocots are the transformation by means of thebiolistic approach (Wan and Lemaux; Plant Physiol. 104 (1994), 37-48;Vasil et al.; Biotechnology 11 (1992), 667-674; Ritala et al., PlantMol. Biol 24, (1994) 317-325; Spencer et al., Theor. Appl. Genet. 79(1990), 625-631), protoplast transformation, the electroporation ofpartially permeabilized cells, and the introduction of DNA by means ofglass fibers. In particular the transformation of maize has beendescribed repeatedly in the literature (WO 95/06128; EP 0513849 A1; EP0465875 A1; EP 0292435 A1; Fromm et al., Biotechnology 8 (1990),833-844; Gordon-Kamm et al., Plant Cell 2 (1990), 603-618; Koziel etal., Biotechnology 11(1993) 194-200; Moroc et al., Theor AppliedGenetics 80 (190) 721-726). The generation of protoplasts andtransformation with the aid of PEG (Wiebe et al. (1997) Mycol. Res. 101(7): 971-877; Proctor et al. (1997) Microbiol. 143, 2538-2591), on theone hand, and transformation with the assistance of Agrobacteriumtumefaciens (de Groot et al. (1998) Nat. Biotech. 16, 839-842), on theother hand, lend themselves to the transformation of filamentous fungi.

The successful transformation of other cereal species has also alreadybeen described for example in the case of barley (Wan and Lemaux, seeabove; Ritala et al., see above, and wheat (Nehra et al., Plant J.5(1994) 285-297).

Agrobacteria which have been transformed with a vector according to theinvention can likewise be used in a known manner for the transformationof plants, such as test plants like Arabidopsis or crop plants likecereals, maize, oats, rye, barley, wheat, soya, rice, cotton, sugarbeet,canola, sunflower, flax, hemp, potato, tobacco, tomato, carrot,capsicum, oilseed rape, tapioca, cassaya, arrowroot, Tagetes, alfalfa,lettuce and the various tree, nut and grapevine species, for example bybathing scarified leaves or leaf segments in an agrobacterial solutionand subsequently growing them in suitable media.

The genetically modified plant cells can be regenerated via all methodswith which the skilled worker is familiar. Such methods can be found inthe abovementioned publications by S. D. Kung and R. Wu, Potrykus orHöfgen and Willmitzer.

The transgenic organisms generated by transformation with one of theabove-described embodiments of an expression cassette comprising anucleic acid sequence according to the invention or a vector comprisingthe abovementioned expression cassette, and the recombinant NCR whichcan be obtained from the transgenic organism by means of expression, aresubject matter of the present invention. The use of transgenic organismscomprising an expression cassette according to the invention, forexample for providing recombinant protein, and/or the use of theseorganisms in in-vivo assay systems are likewise subject matter of thepresent invention.

Preferred organisms for the recombinant expression are not onlybacteria, yeasts, mosses, algae and fungi, but also eukaryotic celllines.

Preferred mosses are Physcomitrella patens or other mosses described inKryptogamen [cryptogams], Vol. 2, Moose, Farne [mosses, ferns], 1991,Springer Verlag (ISBN 3540536515).

Preferred within the bacteria are, for example, bacteria from the genusEscherichia, Erwinia, Flavobacterium, Alcaligenes or cyanobacteria, forexample from the genus Synechocystis or Anabena.

Preferred yeasts are Candida, Saccharomyces, Schizosaccharomyces,Hansenula or Pichia.

Preferred fungi are Aspergillus, Trichoderma, Ashbya, Neurospora,Fusarium, Beauveria, Mortierella, Saprolegnia, Pythium, or other fungidescribed in Indian Chem Engr. Section B. Vol 37, No 1,2 (1995).

Preferred plants are selected in particular among monocotyledonous cropplants such as, for example, cereal species such as wheat, barley,sorghum or millet, rye, triticale, maize, rice or oats, and sugar cane.The transgenic plants according to the invention are, furthermore, inparticular selected from among dicotyledonous crop plants such as, forexample, Brassicaceae such as oilseed rape, cress, Arabidopsis, cabbagesor canola; Leguminosae such as soya, alfalfa, pea, beans or peanut,Solanaceae such as potato, tobacco, tomato, eggplant or capsicum;steraceae such as sunflower, Tagetes, lettuce or Calendula;Cucurbitaceae such as melon, pumpkin/squash or zucchini, or linseed,cotton, hemp, flax, red pepper, carrot, sugar beet, or various tree, nutand grapevine species.

In principle, transgenic animals such as, for example, C. elegans, arealso suitable as host organisms.

Also preferred is the use of expression systems and vectors which areavailable to the public or commercially available.

Those which must be mentioned for use in E. coli bacteria are thetypical advantageous commercially available fusion and expressionvectors pGEX [Pharmacia Biotech Inc; Smith, D. B. and Johnson, K. S.(1988) Gene 67:31-40], pMAL (New England Biolabs, Beverly, Mass.) andpRIT5 (Pharmacia, Piscataway, N.J.), which contains glutathione Stransferase (GST), maltose binding protein or protein A, the pTrcvectors (Amann et al., (1988) Gene 69:301-315), “pKK233-2” by CLONTECH,Palo Alto, Calif. and the “pET” and “pBAD” vector series fromStratagene, La Jolla.

Further advantageous vectors for use in yeast are pYepSec1 (Baldari, etal., (1987) Embo J. 6:229-234), pMFa (Kurjan and Herskowitz, (1982) Cell30:933-943), pJRY88 (Schultz et al., (1987) Gene 54:113-123), and pYESderivatives, pGAPZ derivatives, pPICZ derivatives, and the vectors ofthe “Pichia Expression Kit” (Invitrogen Corporation, San Diego, Calif.).Vectors for use in filamentous fungi are described in: van den Hondel,C. A. M. J. J. & Punt, P. J. (1991) “Gene transfer systems and vectordevelopment for filamentous fungi, in: Applied-Molecular Genetics ofFungi, J. F. Peberdy, et al., eds., p. 1-28, Cambridge University Press:Cambridge.

As an alternative, insect cell expression vectors may also be usedadvantageously, for example for expression in Sf9, Sf21 or Hi5 cells,which are infected via recombinant baculoviruses. Examples of these arethe vectors of the pAc series (Smith et al. (1983) Mol. Cell Biol.3:2156-2165) and the pVL series (Lucklow and Summers (1989) Virology170:31-39). Others which may be mentioned are the baculovirus expressionsystems “MaxBac 2.0 Kit” and “Insect Select System” by Invitrogen,Carlsbad or “BacPAK Baculovirus Expression System” by CLONTECH, PaloAlto, Calif. Insect cells are particularly suitable for overexpressingeukaryotic proteins since they effect posttranslational modifications ofthe proteins which are not possible in bacteria and yeasts. The skilledworker is familiar with the handling of cultured insect cells and withtheir infection for expressing proteins, which can be carried outanalogously to known methods (Luckow and Summers, Bio/Tech. 6, 1988, pp.47-55; Glover and Hames (eds) in DNA Cloning 2, A practical Approach,Expression Systems, Second Edition, Oxford University Press, 1995,205-244).

Plant cells or algal cells are others which can be used advantageouslyfor expressing genes. Examples of plant expression vectors can be foundas mentioned above in Becker, D., et al. (1992) “New plant binaryvectors with selectable markers located proximal to the left border”,Plant Mol. Biol. 20: 1195-1197 or in Bevan, M. W. (1984) “BinaryAgrobacterium vectors for plant transformation”, Nucl. Acid. Res. 12:8711-8721.

Moreover, the nucleic acid sequences according to the invention can beexpressed in mammalian cells. Examples of suitable expression vectorsare pCDM8 and pMT2PC, which are mentioned in: Seed, B. (1987) Nature329:840 or Kaufman et al. (1987) EMBO J. 6:187-195). Promoterspreferably to be used in this context are of viral origin such as, forexample, promoters of polyoma virus, adenovirus 2, cytomegalovirus orsimian virus 40. Further prokaryotic and eukaryotic expression systemsare mentioned in Chapter 16 and 17 in Sambrook et al., MolecularCloning: A Laboratory Manual. 2nd ed., Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.Further advantageous vectors are described in Hellens et al. (Trends inplant science, 5, 2000).

The organisms transformed with an expression cassette according to theinvention come under the term “transgenic organism according to theinvention”.

The present invention furthermore relates to the use of NCR in a methodfor identifying herbicidally active compounds.

The method according to the invention for identifying herbicidallyactive compounds preferably comprises the following steps:

-   i. bringing an NCR into contact with one or more test compounds    under conditions which permit binding of the test compound(s) to the    NCR; and-   ii. detecting whether the test compound binds to the NCR of i); or-   iii. detecting whether the test compound reduces or blocks the    activity of the NCR of i); or-   iv. detecting whether the test compound reduces or blocks the    transcription, translation or expression of the NCR of i).

The detection in accordance with step (ii) of the above method can beeffected using techniques which identify the interaction between proteinand ligand. In this context, either the test compound or the enzyme cancontain a detectable label such as, for example, a fluorescent label, aradioisotope, a chemiluminescent label or an enzyme label. Examples ofenzyme labels are horseradish peroxidase, alkaline phosphatase orluciferase. The subsequent detection depends on the label and is knownto the skilled worker.

In this context, five preferred embodiments which are also suitable forhigh-throughput screening methods (HTS) in connection with the presentinvention, must be mentioned in particular:

-   1. The average diffusion rate of a fluorescent molecule as a    function of the mass can be determined in a small sample volume via    fluorescence correlation spectroscopy (FCS) (Proc. Natl. Acad. Sci.    USA (1994) 11753-11575). FCS can be employed for determining    protein/ligand interactions by measuring the changes in the mass, or    the changed diffusion rate which this entails, of a test compound    when binding to NCR. A method according to the invention can be    designed directly for measuring the binding of a test compound    labeled with a fluorescent molecule. As an alternative, the method    according to the invention can be designed in such a way that a    chemical reference compound which is labeled with a fluorescent    molecule is displaced by further test compounds (“displacement    assay”). The compounds which are identified in this manner may be    suitable as inhibitors.-   2. Fluoresence polarization exploits the characteristic of a    quiescent fluorophore excited with polarized light to likewise emit    polarized light. If, however, the fluorophore is allowed to rotate    during the excited state, the polarization of the fluorescent light    which is emitted is more or less lost. Under otherwise identical    conditions (for example temperature, viscosity, solvent), the    rotation is a function of molecule size, whereby findings regarding    the size of the fluorophore-bound residue can be obtained via the    reading (Methods in Enzymology 246 (1995), pp. 283-300). A method    according to the invention can be designed directly for measuring    the binding of a fluorescently labeled test compound to the NCR. As    an alternative, the method according to the invention may also take    the form of the “displacement assay” described under 1. The    compounds identified in this manner may be suitable as inhibitors.-   3. Fluorescent resonance energy transfer (FRET) is based on the    irradiation-free energy transfer between two spatially adjacent    fluorescent molecules under suitable conditions. A prerequisite is    that the emission spectrum of the donor molecule overlaps with the    excitation spectrum of the acceptor molecule. By labelling NCR with    a fluorescent label and the binding test compound, the binding can    be measured by means of FRET (Cytometry 34, 1998, pp. 159-179). As    an alternative, the method according to the invention may also take    the form of the “displacement assay” described under 1. An    especially suitable embodiment of FRET technology is “Homogeneous    Time Resolved Fluorescence” (HTRF) as can be obtained from Packard    BioScience. The compounds which are identified in this manner may be    suitable as inhibitors.-   4. Surface-enhanced laser desorption/ionization (SELDI) in    combination with a time-of-flight mass spectrometer (MALDI-TOF)    makes possible the rapid analysis of molecules on a support and can    be used for analyzing protein/ligand interactions (Worral et    al., (1998) Anal. Biochem. 70:750-756). In a preferred embodiment,    NCR is immobilized on a suitable support and incubated with the test    compound. After one or more suitable wash steps, the test compound    molecules which are additionally bound to NCR can be detected by    means of the above-mentioned methodology and inhibitors can thus be    selected. The compounds which are identified in this manner may be    suitable as inhibitors.-   5. The measurement of surface plasmon resonance is based on the    change in the refractive index at a surface when a test compound    binds to a protein which is immobilized to said surface. Since the    change in the refractive index is identical for virtually all    proteins and polypeptides for a defined change in the mass    concentration at the surface, this method can be applied to any    protein in principle (Lindberg et al. Sensor Actuators 4 (1983)    299-304; Malmquist Nature 361 (1993) 186-187). The measurement can    be carried out for example with the automatic analyzer based on    surface plasmon resonance which is available from Biacore (Freiburg)    at a throughput of, currently, up to 384 samples per day. A method    according to the invention can be designed directly for measuring    the binding of a test compound to NCR. As an alternative, the method    according to the invention may also take the form of the    “displacement assay” described under 1. The compounds identified in    this manner may be suitable as inhibitors.

All of the substances identified via the abovementioned methods cansubsequently be checked for their herbicidal action in anotherembodiment of the method according to the invention.

Furthermore, there exists the possibility of detecting furthercandidates for herbicidal active ingredients by molecular modeling viaelucidation of the three-dimensional structure of NCR by x-ray structureanalysis. The preparation of protein crystals required for x-raystructure analysis, and the relevant measurements and subsequentevaluations of these measurements, the detection of a binding site inthe protein, and the prediction of potential inhibitor structures areknown to the skilled worker. In principle, an optimization of thecompounds identified by the abovementioned methods is also possible viamolecular modeling.

A preferred embodiment of the method according to the invention, whichis based on steps i) and ii), consists in

-   i. expressing an NCR in a transgenic organism according to the    invention, or growing an organism which naturally contains an NCR;-   ii. bringing the NCR of step i) in the cell digest of the transgenic    or nontransgenic organism, in partially purified form or in    homogeneously purified form, into contact with a test compound; and-   iii. selecting a compound which reduces or blocks the NCR activity,    the activity of the NCR incubated with the test compound being    compared with the activity of an NCR not incubated with a test    compound.

The solution containing the NCR can consist of the lysate of theoriginal organism or of the transgenic organism which has beentransformed with an expression cassette according to the invention. Ifappropriate, the NCR can be purified partially or fully via customarymethods. A general overview over current protein purification techniquesis described, for example, in Ausubel, F. M. et al., Current Protocolsin Molecular Biology, Greene Publishing Assoc. and Wiley-Interscience(1994); ISBN 0-87969-309-6. If NCR is obtained recombinantly, theprotein which takes the form of a fusion with an affinity tag can bepurified via affinity chromatography as is known to the skilled worker.NCR from human tissue may be purified, for example, by the method ofJollie et al. (Plant Physiol. 85, pp 457-462, 1987).

The NCR which is required for in-vitro methods can thus be isolatedeither by means of heterologous expression from a transgenic organismaccording to the invention or from an organism containing NCR activity,preferably from an undesired plant, the term “undesired plant” beingunderstood as meaning the species mentioned at the outset.

To identify herbicidal compounds, NCR is incubated with a test compound.After a reaction time, the activity of an NCR incubated with the testcompound is determined with the activity of an NCR not incubated withthe test compound. If the NCR is inhibited, a significant decrease inactivity in comparison with the activity of the noninhibited polypeptideaccording to the invention is observed, the result being a reduction ofat least 10%, advantageously at least 20%, preferably at least 30%,especially preferably by at least 50%, up to 100% reduction (blocking).Preferred is an inhibition of at least 50% at test compoundconcentrations of 10⁻⁴ M, preferably at 10⁻⁵ M, especially preferably of10⁻⁶ M, based on enzyme concentration in the micromolar range.

The enzyme activity of NCR can be determined for example by means of anactivity assay in which the increase in the product, the decrease in thesubstrate (or starting material) or the decrease in a specific cofactorare determined as a function of a defined period of time, or by acombination of at least two of the abovementioned parameters.

Examples of suitable substrates are, for example, iron(III) cytochromeb5, potassium iron(III) cyanide, 2,6-dichlorophenolindophenol,methemerythrin, p-benzoquinone or 5-hydroxy-1,4-naphthoquinone,preferably iron(III) cytochrome b5, potassium iron(III) cyanide,2,6-dichlorophenolindophenol, especially preferably iron(III) cytochromeb5, potassium iron(III) cyanide, very especially preferably potassiumiron(III) cyanide and, for suitable cofactors, NADH. If appropriate,derivatives of the abovementioned compounds which contain a detectablelabel, such as, for example, a fluorescent label, a radioisotope or achemiluminescent label, may also be used.

The amounts of substrate to be employed in the activity assay range from0.5-10 mM, and the amounts of NADH range from 0.1-5 mM, based on 1-100μg/ml enzyme.

In an especially preferred embodiment, the conversion of a substrate ismonitored photometrically, using a modification of a method described byMihara and Sato (Methods Enzymol., 52, 1978, pp. 102-108) which is basedon the reduction of potassium iron(III) cyanide and photometricmeasurement at 420 nm.

A preferred embodiment of the method according to the invention which isbased on steps i) and iii) consists of the following steps:

-   i. generation of a transgenic organism according to the invention;-   ii. applying a test compound to the transgenic organism of i) and to    a nontransgenic organism of the same genotype;-   iii. determining the growth or the viability of the transgenic and    the nontransgenic organisms after application of the test compound;    and-   iv. selection of test compounds which bring about a reduced growth    or a reduced viability of the nontransgenic organism in comparison    with the growth of the transgenic organism.

In this method, the polypeptide with the biological activity of an NCRis overexpressed in the transgenic organism of i). The transgenicorganism thus shows an elevated NCR activity in comparison with anontransgenic organism, elevated NCR activity of the transgenic organismbeing understood as meaning an activity which exceeds the activity ofthe nontransgenic organism of the same genus by at least 10%, preferablyby at least 25%, especially preferably by at least 40%, very especiallypreferably by at least 50%.

In this context, the difference in growth in step iv) for the selectionof a herbicidally active inhibitor amounts to at least 10%, preferably20%, by preference 30%, especially preferably 40% and very especiallypreferably 50%.

The transgenic organism in this context is a bacterium, a yeast, afungus, a plant or a eukaryotic cell line (derived from insects or frommammals such as, for example, mice), preferably plants, bacteria oryeasts, which can readily be transformed by means of customarytechniques, such as Arabidopsis thaliana, Solanum tuberosum, Nicotianatabacum or Saccharomyces cerevisiae, into which the sequence encoding apolypeptide according to the invention has been incorporated bytransformation. These transgenic organisms thus show increased toleranceto compounds which inhibit the polypeptide according to the invention.Saccharomyces cerevisiae is in particular the organism of choice sinceits genome has been sequenced in its entirety and it can readily be usedfor the generation of “knock-out” mutants (for example Methods in YeastGenetics, Kaiser, Michaelis, Mitchell (eds.) CSHL Press, Cold SpringHarbor Laboratory Press, 1994: 73-85) and the analogous NCR gene whichis present in this organism can be silenced in a targeted manner.

However, the abovementioned method can also be used for identifyingcompounds with a growth-regulatory action. In this context, thetransgenic organism employed is a plant. The method for identifyinggrowth-regulatory compounds thus encompasses the following steps:

-   i. generation of a transgenic plant comprising a nucleic acid    sequence according to the invention encoding an NCR;-   ii. applying a test substance to the transgenic plant of i) and to a    nontransgenic plant of the same variety;-   iii. determining the growth or the viability of the transgenic and    the nontransgenic plants after application of the test substance;    and-   iv. selection of test substances which bring about modified growth    of the nontransgenic plant in comparison with the growth of the    transgenic plant.

Step iv) involves the selection of test compounds which bring about amodified growth of the nontransgenic organism in comparison with thegrowth of the transgenic organism. Modified growth is understood asmeaning, in this context, inhibition of the vegetative growth of theplants, which can manifest itself in particular in reduced longitudinalgrowth. Accordingly, the treated plants show stunted growth; moreover,their leaves are darker. In addition, modified growth is also understoodas meaning a change of the course of maturation over time, theinhibition or promotion of lateral branched growth of the plants,shortened or extended developmental stages, increased standing ability,the growth of larger amounts of buds, flowers, leaves, fruits, seedkernels, roots and tubers, an increased sugar content in plants such assugarbeet, sugar cane and citrus fruit, an increased protein content inplants such as cereals or soya, or stimulation of the latex flow inrubber trees. The skilled worker is familiar with the detection of suchmodified growth.

It is also possible, in the method according to the invention, to employa plurality of test compounds in a method according to the invention. Ifa group of test compounds affects the target, then it is either possibledirectly to isolate the individual test compounds or to divide the groupof test compounds into a variety of subgroups, for example when itconsists of a multiplicity of different components, in order to reducethe number of the different test compounds in the method according tothe invention. The method according to the invention is then repeatedwith the individual test compound or the relevant subgroup of testcompounds. Depending on the complexity of the sample, theabove-described steps can be carried out repeatedly, preferably untilthe subgroup identified in accordance with the method according to theinvention only comprises a small number of test compounds, or indeedjust one test compound.

All of the above-described methods for identifying herbicidally activeinhibitors are hereinbelow referred to as “methods according to theinvention”.

All of the compounds or substances which have been identified via themethods according to the invention can subsequently be tested in vivofor their herbicidal action. One possibility of testing the compoundsfor herbicidal action is to use duckweed, Lemna minor, in microtiterplates. Parameters which can be measured are modifications in thechlorophyll content and the photosynthesis rate. It is also possible toapply the compound directly to undesired plants, it being possible toidentify the herbicidal action for example via restricted growth.

The method according to the invention can advantageously also be carriedout in high-throughput methods, or high-throughput screening methods(HTS), which enable the simultaneous testing of a multiplicity ofdifferent compounds.

The use of supports which contain one or more of the nucleic acidmolecules according to the invention, one or more of the vectorscontaining the nucleic acid sequence according to the invention, one ormore transgenic organisms containing at least one of the nucleic acidsequences according to the invention or one or more (poly)peptidesencoded by the nucleic acid sequences according to the invention lendsitself to carrying out an HTS in practice. The support used can be solidor liquid, it is preferably solid and especially preferably a microtiterplate. The abovementioned supports are also subject matter of thepresent invention. In accordance with the most widely used technique,96-well, 384-well and 1536-well microtiter plates which, as a rule, cancomprise volumes of 200 μl, are used. Besides the microtiter plates, thefurther components of an HTS system which match the correspondingmicrotiter plates, such as a large number of instruments, materials,automatic pipetting devices, robots, automated plate readers and platewashers, are commercially available.

In addition to the HTS methods which are based on microtiter plates,what are known as free-format assays, or assay systems where there areno physical barriers between the samples, may also be used, as, forexample, in Jayaickreme et al., Proc. Natl. Acad. Sci U.S.A. 19 (1994)161418; Chelsky, “Strategies for Screening Combinatorial Libraries,First Annual Conference of The Society for Biomolecular Screening inPhiladelphia, Pa. (Nov. 710, 1995); Salmon et al., Molecular Diversity 2(1996), 5763 and U.S. Pat. No. 5,976,813.

The invention furthermore relates to herbicidally active compoundsidentified by the methods according to the invention. These compoundsare hereinbelow referred to as “selected compounds”. They have amolecular weight of less than 1000 g/mol, advantageously less than 500g/mol, preferably less than 400 g/mol, especially preferably less than300 g/mol. Herbicidally active compounds have a Ki value of less than 1mM, preferably less than 1 μM, especially preferably less than 0.1 μM,very especially preferably less than 0.01 μM.

Naturally, the selected compounds can also be present in the form oftheir agriculturally useful salts. Agriculturally useful salts which aresuitable are mainly the salts of those cations, or the acid additionsalts of those acids, whose cations, or anions, do not adversely affectthe herbicidal action of the selected compounds.

If the selected compounds contain asymmetrically substituted α-carbonatoms, they may furthermore also be present in the form of racemates,enantiomer mixtures, pure enantiomers or, if they have chiralsubstituents, also in the form of diastereomer mixtures.

The selected compounds can be chemically synthesized substances orsubstances produced by microorganisms and can be found, for example, incell extracts of, for example, plants, animals or microorganisms. Thereaction mixture can be a cell-free extract or comprise a cell or cellculture. Suitable methods are known to the skilled worker and aredescribed generally for example in Alberts, Molecular Biology the cell,3^(rd) Edition (1994), for example Chapter 17. The selected compoundsmay also originate from extensive substance libraries.

Candidate test compounds can be expression libraries such as, forexample, cDNA expression libraries, peptides, proteins, nucleic acids,antibodies, small organic substances, hormones, PNAs or the like(Milner, Nature Medicin 1 (1995), 879-880; Hupp, Cell. 83 (1995),237-245; Gibbs, Cell. 79 (1994), 193-198 and references cited therein).

The selected compounds can be used for controlling undesired vegetation,if appropriate also for the defoliation of, for example, potatoes or thedesiccation of, for example, cotton, and as growth regulators.Herbicidal compositions comprising the selected compounds afford verygood control of vegetation on noncrop areas. In crops such as wheat,rice, maize, soybean and cotton, they act against broad-leaved weeds andgrass weeds without inflicting any significant damage on the cropplants. This effect is observed in particular at low application rates.The selected compounds can be used for controlling the harmful plantswhich have already been mentioned above.

Depending on the application method in question, selected compounds, orherbicidal compositions comprising them, can advantageously also beemployed in a further number of crop plants for eliminating undesiredplants. Examples of suitable crops are:

Allium cepa, Ananas comosus, Arachis hypogaea, Asparagus officinalis,Beta vulgaris spec. altissima, Beta vulgaris spec. rapa, Brassica napusvar. napus, Brassica napus var. napobrassica, Brassica rapa var.silvestris, Camellia sinensis, Carthamus tinctorius, Caryaillinoinensis, Citrus limon, Citrus sinensis, Coffea arabica (Coffeacanephora, Coffea liberica), Cucumis sativus, Cynodon dactylon, Daucuscarota, Elaeis guineensis, Fragaria vesca, Glycine max, Gossypiumhirsutum, (Gossypium arboreum, Gossypium herbaceum, Gossypiumvitifolium), Helianthus annuus, Hevea brasiliensis, Hordeum vulgare,Humulus lupulus, Ipomoea batatas, Juglans regia, Lens culinaris, Linumusitatissimum, Lycopersicon lycopersicum, Malus spec., Manihotesculenta, Medicago sativa, Musa spec., Nicotiana tabacum (N. rustica),Olea europaea, Oryza sativa, Phaseolus lunatus, Phaseolus vulgaris,Picea abies, Pinus spec., Pisum sativum, Prunus avium, Prunus persica,Pyrus communis, Ribes sylestre, Ricinus communis, Saccharum officinarum,Secale cereale, Solanum tuberosum, Sorghum bicolor (s. vulgare),Theobroma cacao, Trifolium pratense, Triticum aestivum, Triticum durum,Vicia faba, Vitis vinifera, Zea mays.

In addition, the selected compounds can also be used in crops whichtolerate the action of herbicides owing to breeding, includingrecombinant methods. The generation of such crops is describedhereinbelow.

The invention furthermore relates to a method of preparing theherbicidal composition which has already been mentioned above, whichcomprises formulating selected compounds with suitable auxiliaries togive crop protection products.

The selected compounds can be formulated for example in the form ofdirectly sprayable aqueous solutions, powders, suspensions, also highlyconcentrated aqueous, oily or other suspensions or suspoemulsions ordispersions, emulsifiable concentrates, emulsions, oil dispersions,pastes, dusts, materials for spreading or granules by means of spraying,atomizing, dusting, spreading or pouring. The use forms depend on theintended use and the nature of the selected compounds; in any case, theyshould guarantee the finest possible distribution of the selectedcompounds. The herbicidal compositions comprise a herbicidally activeamount of at least one selected compound and auxiliaries conventionallyused in the formulation of herbicidal compositions.

For the preparation of emulsions, pastes or aqueous or oily formulationsand dispersible concentrates (DC), the selected compounds can bedissolved or dispersed in an oil or solvent, it being possible to addfurther formulation auxiliaries for homogenization purposes. However, itis also possible to prepare liquid or solid concentrates from selectedcompound, if appropriate solvents or oil and, optionally, furtherauxiliaries, and these concentrates are suitable for dilution withwater. The following can be mentioned: emulsifiable concentrates (EC,EW), suspensions (SC), soluble concentrates (SL), dispersibleconcentrates (DC), pastes, pills, wettable powders or granules, it beingpossible for the solid formulations to be either soluble or dispersible(wettable) in water. In addition, suitable powders or granules ortablets can additionally be provided with a solid coating which preventsabrasion or premature release of the active ingredient.

In principle, the term “auxiliaries” is understood as meaning thefollowing classes of compounds: antifoam agents, thickeners, wetters,stickers, dispersants, emulsifiers, bactericides and/or thixotropicagents. The skilled worker is familiar with the meaning of theabovementioned agents.

SLs, EWs and ECs can be prepared by simply mixing the constituents inquestion; powders can be prepared by mixing or grinding in specifictypes of mills (for example hammer mills). DCs, SCs and SEs are usuallyprepared by wet milling, it being possible to prepare an SE from an SCby addition of an organic phase which may comprise further auxiliariesor selected compounds. The preparation is known. Powders, materials forspreading and dusts can advantageously be prepared by mixing orconcomitantly grinding the active substances together with a solidcarrier. Granules, for example coated granules, impregnated granules andhomogeneous granules, can be prepared by binding the selected compoundsto solid carriers. The skilled worker is familiar with further detailsregarding their preparation, which are mentioned for example in thefollowing publications: U.S. Pat. No. 3,060,084, EP-A 707445 (for liquidconcentrates), Browning, “Agglomeration”, Chemical Engineering, Dec. 4,1967, 147-48, Perry's Chemical Engineer's Handbook, 4th Ed.,McGraw-Hill, New York, 1963, pages 8-57 and et seq. WO 91/13546, U.S.Pat. No. 4,172,714, U.S. Pat. No. 4,144,050, U.S. Pat. No. 3,920,442,U.S. Pat. No. 5,180,587, U.S. Pat. No. 5,232,701, U.S. Pat. No.5,208,030, GB 2,095,558, U.S. Pat. No. 3,299,566, Klingman, Weed Controlas a Science, John Wiley and Sons, Inc., New York, 1961, Hance et al.,Weed Control Handbook, 8th Ed., Blackwell Scientific Publications,Oxford, 1989 and Mollet, H., Grubemann, A., Formulation technology,Wiley VCH Verlag GmbH, Weinheim (Federal Republic of Germany), 2001.

The skilled worker is familiar with a multiplicity of inert liquidand/or solid carriers which are suitable for the formulations accordingto the invention, such as, for example, liquid additives such as mineraloil fractions of medium to high boiling point such as kerosene or dieseloil, furthermore coal tar oils and oils of vegetable or animal origin,aliphatic, cyclic and aromatic hydrocarbons, for example paraffin,tetrahydronaphthalene, alkylated naphthalenes or their derivatives,alkylated benzenes or their derivatives, alcohols such as methanol,ethanol, propanol, butanol and cyclohexanol, ketones such ascyclohexanone, or strongly polar solvents, for example amines such asN-methylpyrrolidone or water.

Examples of solid carriers are mineral earths such as silicas, silicagels, silicates, talc, kaolin, limestone, lime, chalk, bole, loess,clay, dolomite, diatomaceous earth, calcium sulfate, magnesium sulfate,magnesium oxide, ground synthetic materials, fertilizers such asammonium sulfate, ammonium phosphate, ammonium nitrate, ureas andproducts of vegetable origin such as cereal meal, tree bark meal, woodmeal and nutshell meal, cellulose powders or other solid carriers.

The skilled worker is familiar with a multiplicity of surface-activesubstances (surfactants) which are suitable for the formulationsaccording to the invention such as, for example, alkali metal salts,alkaline earth metal salts or ammonium salts of aromatic sulfonic acids,for example lignosulfonic acid, phenolsulfonic acid, naphthalenesulfonicacid and dibutylnaphthalenesulfonic acid, and of fatty acids, of alkyl-and alkylarylsulfonates, of alkyl sulfates, lauryl ether sulfates andfatty alcohol sulfates, and salts of sulfated hexa-, hepta- andoctadecanols and of fatty alcohol glycol ethers, condensates ofsulfonated naphthalene and its derivatives with formaldehyde,condensates of naphthalene or of the naphthalenesulfonic acids withphenol and formaldehyde, polyoxyethylene octylphenol ether, ethoxylatedisooctyl-, octyl- or nonylphenol, alkylphenyl polyglycol ethers,tributylphenyl polyglycol ethers, alkylaryl polyether alcohols,isotridecyl alcohol, fatty alcohol/ethylene oxide condensates,ethoxylated castor oil, polyoxyethylene alkyl ethers or polyoxypropylenealkyl ethers, lauryl alcohol polyglycol ether acetate, sorbitol esters,lignin-sulfite waste liquors or methylcellulose.

The herbicidal compositions, or the selected compounds, can be appliedpre- or post-emergence. If the selected compounds are less welltolerated by certain crop plants, application techniques may be used inwhich the selected compounds are sprayed, with the aid of the sprayingapparatus, in such a way that they come into as little contact, if any,with the leaves of the sensitive crop plants while the selectedcompounds reach the leaves of undesired plants which grow underneath, orthe bare soil surface (post-directed, lay-by).

Depending on the intended aim, the season, the target plants and thegrowth stage, the application rates of selected compounds amount to0.001 to 3.0, preferably 0.01 to 1.0 kg/ha.

Providing the herbicidal target furthermore makes possible a method foridentifying a protein with the biological activity of an NCR which isnot inhibited, or inhibited to a limited extent only, by a herbicidewhich has NCR as its selective action, for example the herbicidallyactive selected compounds. A protein which differs in this way from NCRis hereinbelow referred to as NCR variant, which is encoded by a nucleicacid sequence which

-   i) encodes a polypeptide with the biological activity of an    NADH-dependent cytochrome b5 reductase which is not inhibited by    herbicidally active substances which inhibit NCR and which have been    identified by the abovementioned methods; and-   ii) which comprises a functional equivalent of the nucleic acid    sequence SEQ ID NO:1 with at least 52% identity with SEQ ID NO:1; or    which can be derived by backtranslating the amino acid sequence of a    functional equivalent of SEQ ID NO:2 which has at least 39% identity    with SEQ ID NO:2.

In a preferred embodiment, the abovementioned method for generatingnucleic acid sequences encoding NCR variants of nucleic acid sequencescomprise the following steps:

-   a) expression, in a heterologous system or in a cell-free system, of    the proteins encoded by the abovementioned nucleic acids;-   b) randomized or site-directed mutagenesis of the protein by    modification of the nucleic acid;-   c) measuring the interaction of the modified gene product with the    herbicide;-   d) identification of derivatives of the protein which show less    interaction;-   e) assaying the biological activity of the protein after application    of the herbicide;-   f) selection of the nucleic acid sequences which show a modified    biological activity toward the herbicide.

The functional SEQ ID NO:1 equivalents according to the invention haveat least 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, preferably at least60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69% and 70%, preferably atleast 71%, 72%, 73%, 74%, 75%, 76%, especially preferably at least 77%,78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, veryespecially preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% homology with SEQ ID NO:1.

The functional SEQ ID NO:2 equivalents according to the invention haveat least 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%,51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, preferably at least 60%,61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69% and 70%, preferably at least71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,85%, 86%, especially preferably at least 87%, 88%, 89%, 90%, 91%, 92%,93%, very especially preferably at least 94%, 95%, 96%, 97%, 98%, 99%homology with SEQ ID NO:2.

The sequences which are selected by the above-described method areadvantageously introduced into an organism. The invention thereforefurthermore relates to an organism prepared by this method The organismis preferably a plant, especially preferably one of the above-definedcrop plants.

Thereafter, intact plants are regenerated and the resistance to theselected compound is verified in intact plants.

Modified proteins and/or nucleic acids which are capable of conferring,in plants, resistance to the selected compounds can also be generatedfrom the abovementioned nucleic acid sequences via site-directedmutagenesis; this mutagenesis allows for example the stability and/oractivity of the target protein or the characteristics such as bindingand action of the abovementioned inhibitors according to the inventionto be improved or modified in a highly targeted manner.

A site-directed mutagenesis method in plants which can advantageously beused has been described for example by Zhu et al. (Nature Biotech., Vol.18, May 2000: 555-558).

Moreover, modifications can be obtained via the PCR method described bySpee et al. (Nucleic Acids Research, Vol. 21, No. 3, 1993: 777-78) usingdITP for random mutagenesis, or by the further improved method of Relloset al. (Protein Expr. Purif., 5, 1994: 270-277).

A further possibility of generating these modified proteins and/ornucleic acids is an in-vitro recombination technique for olecularevolution, which has been described by Stemmer et al. (Proc. Natl. Acad.Sci. USA, Vol. 91, 1994; 10747-10751), or the combination of the PCR andrecombination method, which has been described by Moore et al. (NatureBiotechnology Vol. 14, 1996: 458-467).

A further way of mutating proteins is described by Greener et al. inMethods in Molecular Biology (Vol. 57, 1996: 375-385). EP-A-0 909 821describes a method of modifying proteins using the microorganism E. coliXL-1 Red. Upon replication, this microorganism generates mutations inthe introduced nucleic acids and thus leads to a modification of thegenetic information. Advantageous nucleic acids and the proteins encodedby them can be identified readily via isolating the modified nucleicacids or the modified proteins and carrying out resistance testing.After introduction into plants, they can manifest resistance therein andthus lead to resistance to the herbicides.

Further methods of mutagenesis and selection are, for example, methodssuch as the in vivo mutagenesis of seeds or pollen and selection ofresistant alleles in the presence of the inhibitors according to theinvention, followed by the genetic and molecular identification of themodified, resistant allele. Furthermore the mutagenesis and selection ofresistances in cell culture by growing the culture in the presence ofsuccessively increasing concentrations of the inhibitors according tothe invention. In doing so, the increase in the spontaneous mutationrate by chemical/physical mutagenic treatment may be exploited. Asdescribed above, modified genes may also be isolated usingmicroorganisms which have an endogenous or recombinant activity of theproteins encoded by the nucleic acids used in the method according tothe invention, which microorganisms are sensitive to the inhibitorsidentified in accordance with the invention. Growing the microorganismson media with increasing concentrations of inhibitors according to theinvention permits the selection and evolution of resistant variants ofthe targets according to the invention. The frequency of the mutations,in turn, can be increased by mutagenic treatments.

In addition, methods are available for the targeted modification ofnucleic acids (Zhu et al. Proc. Natl. Acad. Sci. USA, Vol. 96, 8768-8773and Beethem et al., Proc. Natl. Acad. Sci. USA, Vol 96, 8774-8778).These methods make it possible to replace, in the proteins, those aminoacids which are of importance for binding inhibitors by functionallyequivalent amino acids which, however, inhibit the binding of theinhibitor.

The invention therefore furthermore relates to a method of generatingnucleic acid sequences which encode gene products with a modifiedbiological activity, the biological activity being modified such that anincreased activity is present. Increased activity is to be understood asmeaning an activity which is increased over the original organism, orover the original gene product, by at least 10%, preferably by at least30%, especially preferably by at least 50%, very especially preferablyby at least 100%. Moreover, the biological activity may have beenmodified such that the substances and/or compositions according to theinvention no longer, or no longer correctly, bind to the nucleic acidsequences and/or the gene products encoded by them. No longer, or nolonger correctly, is to be understood as meaning for the purposes of theinvention that the substances bind by at least 30% less, preferably byat least 50% less, especially preferably by at least 70% less, veryespecially preferably by at least 80% less or not at all to the modifiednucleic acids and/or gene products in comparison with the original geneproduct or the original nucleic acids.

Yet another aspect of the invention therefore relates to a transgenicplant which has been transformed with a nucleic acid sequence whichencodes a gene product with a modified biological activity, or with anucleic acid sequence encoding an NCR variant. Transformation methodsare known to the skilled worker and examples have been given furtherabove.

Genetically modified transgenic plants which are resistant to thesubstances found by the methods according to the invention and/or tocompositions comprising these substances can also be generated bytransformation followed by overexpression of a nucleic acid sequenceaccording to the invention. This is why the invention furthermorerelates to a method for generating transgenic plants which are resistantto substances which have been found by a method according to theinvention, which comprises overexpressing, in these plants, nucleicacids encoding an NCR variant. A similar method is described, forexample, in Lermantova et al., Plant Physiol., 122, 2000: 75-83.

The above-described methods according to the invention for generatingresistant plants make possible the development of novel herbicides whichhave as complete as possible an action which is independent of the plantspecies (what are known as nonselective herbicides), in combination withthe development of useful plants which are resistant to the nonselectiveherbicide. Useful plants which are resistant to nonselective herbicideshave already been described on several occasions. In this context, onecan distinguish between several principles for achieving a resistance:

-   a) generation of resistance in a plant via mutation methods or    recombinant methods by markedly overproducing the protein which acts    as target for the herbicide and by the fact that, owing to the large    excess of the protein which acts as target for the herbicide, the    function exerted by this protein in the cell is even retained after    application of the herbicide.-   b) modification of the plant such that a modified version of the    protein which acts as target of the herbicide is introduced and that    the function of the newly introduced modified protein is not    adversely affected by the herbicide.-   c) modification of the plant such that a novel protein/a novel RNA    is introduced wherein the chemical structure of the protein or of    the nucleic acid, such as of the RNA or the DNA, which structure is    responsible for the herbicidal action of the low-molecular-weight    substance, is modified so that, owing to the modified structure, a    herbicidal action can no longer unfold, that is to say the    interaction of the herbicide with the target can no longer take    place.-   d) the function of the target is replaced by a novel gene which has    been introduced into the plant, and what is known as an “alternative    pathway” is created.-   e) the function of the target is taken over by another gene, or its    gene product, present in the plant.

The skilled worker is familiar with alternative methods for identifyingthe homologous nucleic acids, for example in other plants with similarsequences, such as, for example, using transposons. The presentinvention therefore also relates to the use of alternative insertionmutagenesis methods for the insertion of foreign nucleic acids into thenucleic acid sequences SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5 insequences derived from these sequences on the basis of the genetic codeand/or in their derivatives in other plants.

The transgenic plants are generated with one of the above-describedembodiments of the expression cassette according to the invention bycustomary transformation methods which have likewise been describedabove.

The expression efficiency of the recombinantly expressed NCR can bedetermined for example in vitro by shoot meristem propagation or by agermination test. Moreover, the expression of the NCR gene whose natureand level has been modified, and its effect on the resistance to NCRinhibitors, can be tested on test plants in greenhouse experiments.

The invention is illustrated in greater detail by the examples whichfollow, which are not to be considered as limiting.

General DNA Manipulation and Cloning Methods

Cloning methods such as, for example, restriction cleavages, agarose gelelectrophoresis, purification of DNA fragments, transfer of nucleicacids to nitrocellulose and nylon membranes, linking DNA fragments,transformation of Escherichia coli cells, bacterial cultures andsequence analysis of recombinant DNA were carried out as described bySambrook et al. (1989) (Cold Spring Harbor Laboratory Press: ISBN0-87969-309-6) and Ausubel, F. M. et al., Current Protocols in MolecularBiology, Greene Publishing Assoc. and Wiley-Interscience (1994); ISBN0-87969-309-6.

Molecular-biological standard methods for plants and planttransformation methods are described in Schultz et al., Plant MolecularBiology Manual, Kluwer Academic Publishers (1998), Reither et al.,Methods in Arabidopsis Research, World scientific press (1992) andArabidopsis: A Laboratory Manual (2001), ISBN 0-87969-573-0.

The bacterial strains used hereinbelow (E. coli DH5α, XL-1 blue, XL10Gold, BL21DE(3), JM 109) were obtained from Stratagene, BRL Gibco orInvitrogen, Carlsberg, Calif. The vectors used for cloning were pCR T7CTTOPO, pCR T7/NT TOPO and pCR 2.1 TOPO from Invitrogen and pUC 19 fromAmersham Pharmacia (Freiburg), pBinAR (Höfgen and Willmitzer, PlantScience 66, 1990, 221-230) and pMALc2x (New England Biolabs).

EXAMPLE 1 Generation of an Arabidopsis Plant Transformation Vector

The primer pair Rei 156/Rei 157 (SEQ ID NO:5) Rei 156:5′-TATACCCGGGATGGATACCGAGTTTCTCCGAA-3′ and (SEQ ID NO:6) Rei 157:5′-TATACCCGGGGAACTGGAATTGCATCTCCGGA-3′

was derived from a cDNA sequence encoding Arabidopsis thaliana NCR(Accession Number AB007799; Mizutani, M. and Fukuchi-Mizutani, M.,1997). Thereafter, the primers Rei 156/157 were used in a PCR reactionfor amplifying a cDNA fragment from an Arabidopsis thaliana cDNA library(Stratagene). The PCR was carried out under the conditions stated inTable 1. TABLE 1 Temperature [° C.] Time [sec] Number or cycles 95 300 195 60 34 58 90 72 300 72 600 1

Following purification via agarose gel electrophoresis, the resultingfragment (SEQ ID NO:1) was cloned into the vector pPCRScript(pPCRScript-NCR), following the manufacture's instructions. Sequencingallowed the verification of the identity of the full-length ArabidopsisNCR cDNA clone.

The vector pBinAR (Höfgen and Willmitzer 1990, Plant Science 66,221-230) was cleaved using SmaI and ligated to the NCR fragment isolatedfrom the vector pPCRScript-NCR, which had been isolated via SmaI(pBinAR-NCR). The ligation was transformed into XL10 Gold E. coli cellsand pBinAR-NCR-containing clones were identified using adigoxigenin-labeled NCR probe (Roche) with the aid of anti-digoxigeninantibodies. The antisense orientation of the NCR-cDNA in pBinAR-NCR wasconfirmed by sequencing and using PCR reactions with 2 different primerpairs (Rei 143 and Rei 196, and Rei 144 and Rei 195, respectively) underthe conditions stated in Table 2. TABLE 2 Temperature [° C.] Time [sec]Number of cycles 95 120 1 95 60 34 58 60 72 120 72 300 1

The antisense orientation of NCR was confirmed using the primers Rei143: 5′-GCTATGACCATGATTACGCC-3′ (SEQ ID NO:7) and Rei 196:5′-TGAGACATCCGTCCTTGC-3′ (SEQ ID NO:8)

via the appearance of a 740 bp DNA fragment and with the primers Rei144: 5′-ACGTTGTAAAACGACGGCCA-3′ (SEQ ID NO:9) and Rei 195:5′-CCGACTACGTTAGACTCTG-3′ (SEQ ID NO:10)via the appearance of an 885 bp DNA fragment.

EXAMPLE 2 Transformation and Analysis of Arabidopsis Plants

The construct pBinAR-NCR was transformed into the agrobacterial strainpGV 2260. A positively transformed agrobacterial colony was employed fortransforming Arabidopsis plants. The detection of the presence of theconstruct pBinAR-NCR in an agrobacterial colony was via PCR using theprimers Rei 156 and Rei 157 via PCR under the conditions stated in Table2. The DNA template used was an amount of an agrobacterial colony whichwas taken directly from the agar plate.

A 4 ml culture on LB medium (LB medium: 10 g/l peptone, 5 g/l yeastextract, 10 g/l NaCl; pH 7.0; 80 mg/l kanamycin/l and 25 mg/lrifampicin) was inoculated by a single colony of positively transformedagrobacteria on a plate and the culture was incubated overnight at 28°C. Thereafter, a 400 ml culture in LB medium (LB medium supplementedwith 80 mg kanamycin/ml and 25 mg/ml rifampicin) was inoculated withthis culture. After incubation for 12 hours at 28° C. and 220 rpm, theculture was precipitated (8.000 rpm, 20 min.) and resuspended intransformation medium (½ MS medium as described by Murashige T. andSkoog F. 1962. Physiologia Plantarum. 15: 473-497; Owen H. R. and MillerA. R. 1992. Plant Cell, Tissue and Organ Culture 28: 147-150; 0.5 g/l2-(N-morpholino)ethanesulfonic acid), pH 5.8; 50 g/l sucrose). FloweringArabidopsis plants were immersed in the resulting suspensionapproximately 5 times on three occasions at 2.5 day intervals andsubsequently transferred to pots containing moist soil. After incubationfor 6 weeks under long-day conditions in controlled-environment cabinets(daytime temperature 22-24° C., night time temperature 19° C.; relativeatmospheric humidity 65%), the seeds of the plant were harvested.

EXAMPLE 3 Analysis of the Transgenic Plants

To carry out the analysis, seeds of the transformed plants of Example 2were on agar selection plates (2.15 g/l Murashige+Skoog micro and macroelements (Fa. DUCHEFA; as described by Murashige T. and Skoog F. 1962.Physiologia Plantarum. 15: 473-497; Owen H. R.; Miller A. R. 1992. PlantCell, Tissue and Organ Culture 28: 147-150) 0.1 g/l myo-inositol, 0.5g/l MES, 10 g/l sucrose, pH 5.7, 1 ml % by weight vitamin B5, 50 μg/lkanamycin; 15 g/l agar agar).

Plants which have grown on the selection plates were transferred intosoil after 3-4 weeks and incubated for 4-8 weeks incontrolled-environment cabinets under long-day conditions (daytimetemperature 22-24° C., night time temperature 19° C.; relativeatmospheric humidity 65%). After 6 weeks, the seeds were harvested. Theintegration of the antisense NCR gene into the genome of the transgenicplants was verified by PCR under the conditions stated in Table 3. TABLE3 Temperature [° C.] Time [sec] Number of cycles 95 120 1 95 60 34 45-5060 72 120 72 300 1

The template used was genomic DNA (isolation by means of the “DNeasyPlant Mini” kits from QIAGEN, following the manufacturer's instructions)which had been isolated from leaf material of the transgenic lines inquestion. The NCR antisense pBinAR construct used for the transformationwas used as positive control. By deliberately choosing the primers, boththe intrinsic genomic gene, which contains an intron and is thus longerthan the antisense NCR cDNA and the antisense NCR cDNA itself weredetected. In the presence of the antisense NCR cDNA in the genome of thetransgenic plants, the expected fragment lengths for the genomic NCR(with an intron) amounted to approx. 1800 bp when using the primers (SEQID NO:11) Rei 524: 5′-TTCGTTGCTTTCGTCGCCGTT-3′ and (SEQ ID NO:12) Rei525: 5′-GTTTGCAGCCATGGCCTTGTT-3′

and 750 bp for the antisense NCR cDNA when using the primers (SEQ IDNO:11) Rei 524: 5′-TTCGTTGCTTTCGTCGCCGTT-3′ and (SEQ ID NO:13) Rei 527:5′-GGCGGGAAACGACAATCTGATC-3′.

Transgenic plants which contained the construct pBinAR-NCR in antisenseorientation showed substantial accumulation of anthocyanin, that is tosay severely stressed leaves and veins, chlorotic leaves and drasticallyreduced growth. Thus, the fresh weight of transgenic plants (TOgeneration) only amounted to 1-10% of the fresh weight of wild-typeplants after 6 weeks cultivation in the soil under long-day conditionsin controlled-environment cabinets (daytime temperature 22-24° C., nighttime temperature 19° C.; relative atmospheric humidity 65%). The seedswhich developed in the pods of the plants of the transformed TOgeneration were shriveled and did not germinate in 100% of all cases.

Thus, it was, surprisingly, demonstrated for the first time that thenatural expression of NCR encoding sequences is essential for plants andthat reduced expression leads to damage as stated for the abovementionedphenotypes. Thus, it was demonstrated that NCR is suitable as a targetfor herbicides.

EXAMPLE 4 Expression in E. coli

To generate active protein with plant NCR activity, an Arabidopsis cDNAencoding an NCR (Genbank Accession Number: AB007799) was overexpressedin E. coli bacteria. To this end, the NCR-encoding nucleic acid sequencewas amplified via PCR (for example as described by Sambrook, J. et al.(1989) “Molecular cloning: A laboratory manual”, Cold Spring HarborLaboratory Press; 34 cycles; annealing temperature 60° C.;polymerization time 2 min) under standard conditions using pBinAR-NCR astemplate and the primers (SEQ ID NO:14) Rei 153: 5′-TATAGAATTCATGGATACCGAGTTTCTCCGAA-3′ (EcoRI) (SEQ ID NO:15) Rei 483: 5′-TATACTGCAGTCAGAACTGGAATTGCATCTCCGG-3′ (PstI)comprising the restriction enzyme cleavage sites EcoRI and PstI andcloned into the vector pMAL-c2x (New England Biolabs) via therestriction enzyme cleavage sites EcoRI and PstI (pMAL-c2x-NCR).

The pMAL-c2x-NCR constructs were transformed into the E. coli strainJM109 (Stratagene) and NCR was expressed following the manufacturer'sinstructions via IPTG in the form of a fusion protein with maltosebinding protein (NCR-MBP). The protein was purified via affinitychromatography using a maltose column as described by the manufacturerNew England Biolabs.

EXAMPLE 5 In Vitro Assay Systems

The NCR activity was determined by the method of Mihara and Sato(Methods Enzymol., 52, 1978, pp. 102-108) with NCR which had beenexpressed recombinantly as described in Example 4 (Fukuchi-Mizutani etal., Plant Physiol. 119, pp. 353-361, 1999) or by the method describedby Jollie et al. (Plant Physiol. 85, pp. 457-462, 1987).

When following the method of Mihara and Sato (Methods Enzymol., 52,1978, pp. 102-108), 1-10 μg of purified NCR-MBP protein are treated with1 mM potassium iron(III) cyanide in 100 μl of buffer (100 mMK₂HPO—/KH₂PO₄ buffer (mixture of equal parts). The reaction is startedby addition of 0.3 mM β-NADH.

The reduction of potassium iron(III) cyanide is measured photometricallyat 420 nm and 25° C. over a period of 5 to 15 minutes.

EXAMPLE 6 Identification of a Functional Analog from Tobacco

To generate a cDNA library (hereinbelow referred to as binary cDNAlibrary”) in a vector which can be used directly for the transformationof plants, mRNA was isolated from various plant tissues and transcribedinto double-stranded cDNA using the TimeSaver cDNA synthesis kit(Amersham Pharmacia Biotech, Freiburg). The cDNA first-strand synthesiswas carried out using T₁₂₋₁₈ oligonucleotides, following themanufacturer's instructions. After size fractionation and ligation ofEcoRI-NotI adapters following the manufacturer's instructions andfilling up the overhangs with Pfu DNA Polymerase (Stratagene), the cDNApopulation was normalized. This was done following the method of Kohciet al., 1995, Plant Journal 8, 771-776, the cDNA being amplified by PCRwith the oligonucleotide N1 under the conditions stated in Table 4.TABLE 4 Temperature [° C.] Time [sec] Number of cycles 94 300 1 94 8 1052 60 72 180 94 8 10 50 60 72 180 94 8 10 48 60 72 180 72 420 1

The resulting PCR product was bound to the column matrix of the PCRPurification Kit (Qiagen, Hilden) and eluted with 300 mM NaP buffer, pH7.0, 0.5 mM EDTA, 0.04% SDS. The DNA was denatured for 5 minutes in aboiling water bath and subsequently renatured for 24 hours at 60° C. 50μl of the DNA were applied to a hydroxyapatite column and the column waswashed 3 times with 1 ml of 10 mM NaP buffer, pH 6.8. The boundsingle-stranded DNA was eluted with 130 mM NaP buffer, pH 6.8,precipitated with ethanol and dissolved in 40 μl of water. 20 μl of thiswas used for a further PCR amplification as described above. Afterfurther ssDNA concentration, a third PCR amplification was carried outas described above.

The plant transformation vector for taking up the cDNA population whichhad been generated as described above was generated via restrictionenzyme cleavage of the vector pUC18 with SbfI and BamHI, purification ofthe vector fragment followed by filling up of the overhangs with Pfu DNApolymerase and religation with T4 DNA ligase (Stratagene). The resultingconstruct is hereinbelow termed pUC18SbfI-.

The vector pBinAR was first cleaved with NotI, the ends were filled upand the vector was religated, cleaved with SbfI, the ends were filled upand the vector was religated and subsequently cleaved with EcoRI andHindIII. The resulting fragment was ligated into a derivative of thebinary plant transformation vector pPZP (Hajdukiewicz, P, Svab, Z,Maliga, P., (1994) Plant Mol Biol 25:989-994) which enables thetransformation of plants by means of agrobacterium and mediateskanamycin resistance in transgenic plants. The construct generated thusis hereinbelow termed pSun12/35S.

pUC18SbfI- was used as template in a polymerase chain reaction (PCR)with the oligonucleotides V1 and V2 (see Table 3) and Pfu DNApolymerase. The resulting fragment was ligated into the SmaI-cutpSun12/35S, giving rise to pSunblues2. Following cleavage with NotI,dephosphorylation with shrimp alkaline phosphatase (Roche Diagnostics,Mannheim) and purification of the vector fragment, pSunblues2 wasligated with the normalized, likewise NotI-cut cDNA population.Following transformation into

E. coli Xl-1blue (Stratagene), the resulting clones were deposited intomicrotiter plates. The binary cDNA library contains cDNAs in “sense” andin “antisense” orientation under the control of the cauliflower mosaicvirus 35S promoter, and, after transformation into tobacco plants, thesecDNAs can, accordingly, lead to “cosuppression” and “antisense” effects.TABLE 3 Oligonucleotides used Oligonucleotide Nucleic acid sequence N15′-AGAATTCGCGGCCGCT-3′ (SEQ ID NO:16) V1 (PWL93not)5′-CTCATGCGGCCGCGCGCAACGCAATTAAT (SEQ ID NO:17) GTG-3′ V2 (pWL92)5′-TCATGCGGCCGCGAGATCCAGTTCGATGT (SEQ ID NO:18) AAC-3′ G1 (35S)5′-GTGGATTGATGTGATATCTCC-3′ (SEQ ID NO:19) G2 (OCS)5′-GTAAGGATCTGAGCTACACAT-3′ (SEQ ID NO:20)

An NCR-encoding sequence was identified via a digoxygenin-labeled probegenerated using the DIG DNA Labeling Mix (Roche, Mannheim) following themanufacturer's instructions, the plasmid pMAL-c2x-NCR being amplifiedunder standard conditions by PCR with the primers (SEQ ID NO:21) Rei111: 5′-ATGGATACCGAGTTTCTCCGAA-3′ and (SEQ ID NO:22) Rei 222:5′-AACTGGAATTGCATCTCCGGA-3′.

The probe generated thus was used for screening the Nicotiana tabacumcDNA library. The cDNA library was plated using a titer of 2.5×10⁵plaque-forming units and analyzed with the aid of the plaque screeningmethod (T. Maniatis, E. F. Fritsch and J. Sambrook, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y. 1989). 12 phage populations were isolated and used in a secondscreening step, whereby genetically uniform phage populations wereisolated which were used for the in-vivo excision. Restriction analysisrevealed no differences between the cDNA clones, and four clones withthe largest insertions were selected for sequencing. The sequence dataof these clones gave SEQ ID NO:3, which has 77% identity with SEQ IDNO:1.

1. The use of a polypeptide with the biological activity of anNADH-dependent cytochrome b5 reductase encoded by a nucleic acidsequence consisting of a) a nucleic acid sequence with the nucleic acidsequence shown in SEQ ID NO:1; or b) a nucleic acid sequence which, onthe basis of the degeneracy of the genetic code, can be deduced from theamino acid sequence shown in SEQ ID NO:2 by back translation; or c) anucleic acid sequence which, on the basis of the degeneracy of thegenetic code, can be deduced from the amino acid sequence of afunctional equivalent of SEQ ID NO:2, which has at least 39% identitywith SEQ ID NO:2, by back translation; or d) a functional equivalent ofthe nucleic acid sequence SEQ ID NO:1 with at least 52% identity withSEQ ID NO:1, as target for identifying herbicidally active compounds. 2.A plant nucleic acid sequence encoding a polypeptide with the biologicalactivity of an NADH-dependent cytochrome b5 reductase comprising apart-region encompassing: a) a nucleic acid sequence with the nucleicacid sequence shown in SEQ ID NO:3; or b) a nucleic acid sequence which,on the basis of the degeneracy of the genetic code, can be deduced fromthe amino acid sequence shown in SEQ ID NO:4 by back translation; or c)a functional equivalent of the nucleic acid sequence SEQ ID NO:3 with atleast 77% identity with SEQ ID NO:3; d) a nucleic acid sequence which,on the basis of the degeneracy of the genetic code, can be deduced fromthe amino acid sequence of a functional equivalent of SEQ ID NO:4, whichhas at least 87% identity with SEQ ID NO:4, by back translation.
 3. Apolypeptide with the biological activity of an NADH-dependent cytochromeb5 reductase as target for herbicides, encoded by a nucleic acidmolecule as claimed in claim
 2. 4. A method for detecting functionalanalogues of SEQ ID NO:1 a) by preparing a probe and subsequentlyscreening a genomic library or a cDNA library of the species inquestion; or b) by a computer search for analogous sequences inelectronic databases.
 5. An expression cassette comprising a) geneticcontrol sequences in operable linkage with a nucleic acid sequence asclaimed in claim 2; or b) additional functional elements; or c) acombination of a) and b).
 6. A vector comprising an expression cassetteas claimed in claim
 5. 7. A nonhuman transgenic organism comprising atleast one nucleic acid sequence encoding a polypeptide with thebiological activity of an NADH-dependent cytochrome b5 reductase asclaimed in claim 2, an expression cassette as claimed in claim 5 or avector as claimed in claim 6 selected from among bacteria, yeasts,fungi, animal cells or plant cells.
 8. The use of a polypeptide with thebiological activity of an NADH-dependent cytochrome b5 reductase encodedby a nucleic acid sequence consisting of a) a nucleic acid sequence withthe nucleic acid sequence shown in SEQ ID NO:1; or b) a nucleic acidsequence which, on the basis of the degeneracy of the genetic code, canbe deduced from the amino acid sequence shown in SEQ ID NO:2 by backtranslation; or c) a functional equivalent of the nucleic acid sequenceSEQ ID NO:1 with at least 52% identity with SEQ ID NO:1; d) a nucleicacid sequence which, on the basis of the degeneracy of the genetic code,can be deduced from the amino acid sequence of a functional equivalentof SEQ ID NO:2, which has at least 39% identity with SEQ ID NO:2, byback translation, in a method for identifying herbicidally activecompounds.
 9. A method for identifying herbicidally active compounds,encompassing the following steps: i. bringing a polypeptide with thebiological activity of an NADH-dependent cytochrome b5 reductase encodedby a nucleic acid sequence consisting of a) a nucleic acid sequence withthe nucleic acid sequence shown in SEQ ID NO:1; or b) a nucleic acidsequence which, on the basis of the degeneracy of the genetic code, canbe deduced from the amino acid sequence shown in SEQ ID NO:2 by backtranslation; or c) a functional equivalent of the nucleic acid sequenceSEQ ID NO:1 with at least 52% identity with SEQ ID NO:1; or d) a nucleicacid sequence which, on the basis of the degeneracy of the genetic code,can be deduced from the amino acid sequence of a functional equivalentof SEQ ID NO:2, which has at least 39% identity with SEQ ID NO:2, byback translation  into contact with one or more test compounds underconditions which allow the test compound(s) to bind to theNADH-dependent cytochrome b5 reductase; and ii. detecting whether thetest compound binds to the NADH-dependent cytochrome b5 reductase of i);or iii. detecting whether the test compound reduces or blocks theactivity of the NADH-dependent cytochrome b5 reductase of i); or iv.detecting whether the test compound reduces or blocks the transcription,translation or expression of the NADH-dependent cytochrome b5 reductaseof i).
 10. A method as claimed in claim 9, which comprises i. eitherexpressing, in a nonhuman transgenic organism, NADH-dependent cytochromeb5 reductase which is encoded by a nucleic acid sequence consisting ofa) a nucleic acid sequence with the nucleic acid sequence shown in SEQID NO:1; or b) a nucleic acid sequence which, on the basis of thedegeneracy of the genetic code, can be deduced from the amino acidsequence shown in SEQ ID NO:2 by back translation; or c) a functionalequivalent of the nucleic acid sequence SEQ ID NO:1 with at least 52%identity with SEQ ID NO:1; or d) a nucleic acid sequence which, on thebasis of the degeneracy of the genetic code, can be deduced from theamino acid sequence of a functional equivalent of SEQ ID NO:2, which hasat least 39% identity with SEQ ID NO:2, by back translation  orculturing an organism which naturally contains NADH-dependent cytochromeb5 reductase; ii. bringing the NADH-dependent cytochrome b5 reductase ofstep i) in a cell digest of the transgenic or nontransgenic organism, inpartially or homogeneously purified form, into contact with a testcompound; and iii. selecting a test compound which reduces or blocks theactivity of the NADH-dependent cytochrome b5 reductase of step i), wherethe activity of the NADH-dependent cytochrome b5 reductase incubatedwith the test compound is compared with the activity of anNADH-dependent cytochrome b5 reductase which is not incubated with atest compound.
 11. A method as claimed in claim 10, wherein, in stepiii), the activity of the NADH-dependent cytochrome b5 reductase isdetermined by using iron(III) cytochrome b5, potassiumiron(III)-cyanide, 2,6-dichlorophenolindophenol, methemerythrin,p-benzoquinone or 5-hydroxy-1,4-naphthoquinone as the substrate.
 12. Amethod as claimed in claim 9, which encompasses the following steps: i.generating a nonhuman transgenic organism as claimed in claim 7 or atransgenic organism comprising a nucleic acid sequence encoding apolypeptide with the biological activity of an NADH-dependent cytochromeb5 reductase consisting of a) a nucleic acid sequence with the nucleicacid sequence shown in SEQ ID NO:1; or b) a nucleic acid sequence which,on the basis of the degeneracy of the genetic code, can be deduced fromthe amino acid sequence shown in SEQ ID NO:2 by back translation; or c)a functional equivalent of the nucleic acid sequence SEQ ID NO:1 with atleast 52% identity with SEQ ID NO:1; or d) a nucleic acid sequencewhich, on the basis of the degeneracy of the genetic code, can bededuced from the amino acid sequence of a functional equivalent of SEQID NO:2, which has at least 39% identity with SEQ ID NO:2, by backtranslation; ii. applying a test substance to the transgenic organism ofclaim i) and to a nontransgenic organism of the same genotype; iii.determining the growth or the viability of the transgenic and of thenontransgenic organisms after application of the test substance; and iv.selection of test substances which bring about a reduced growth or areduced viability of the nontransgenic organism in comparison with thegrowth of the transgenic organism.
 13. A method as claimed in claim 12,which is carried out in a plant organism or a yeast.
 14. A method foridentifying growth-regulatory compounds, comprising the following steps:i. generation of a transgenic plant comprising a nucleic acid sequenceencoding a polypeptide with the biological activity of an NADH-dependentcytochrome b5 reductase consisting of a) a nucleic acid sequence withthe nucleic acid sequence shown in SEQ ID NO:1; or b) a nucleic acidsequence which, on the basis of the degeneracy of the genetic code, canbe deduced from the amino acid sequence shown in SEQ ID NO:2 by backtranslation; or c) a functional equivalent of the nucleic acid sequenceSEQ ID NO:1 with at least 52% identity with SEQ ID NO:1; or d) a nucleicacid sequence which, on the basis of the degeneracy of the genetic code,can be deduced from the amino acid sequence of a functional equivalentof SEQ ID NO:2, which has at least 39% identity with SEQ ID NO:2, byback translation; where the polypeptide with the biological activity ofan NADH-dependent cytochrome b5 reductase is overexpressed in thetransgenic plant; ii. applying a test substance to the transgenic plantof i) and to a nontransgenic plant of the same variety; iii. determiningthe growth or the viability of the transgenic and of the nontransgenicplants after application of the test substance; and selection of testsubstances which bring about a modified growth of the nontransgenicplant in comparison with the growth of the transgenic plant.
 15. Avehicle which has one or more of the nucleic acid molecules as claimedin any of claims 1 to 2, or one or more expression cassettes as claimedin claim 5, or one or more vectors as claimed in claim 6, or one or moreorganisms as claimed in claim 7 or one or more (poly)peptides as claimedin claim
 3. 16. A method as claimed in any of claims 9 to 14, whereinthe substances are identified in a high-throughput screening.
 17. Aherbicidally active compound identified by one or the methods as claimedin any of claims 9 to 13 and
 16. 18. A growth-regulatory compoundidentified by the method as claimed in claims 14 and
 16. 19. A processfor the preparation of an agrochemical composition, which comprises a)identifying a herbicidally active compound by one or the methods asclaimed in any of claims 9 to 13 and 16 or a growth-regulatory compoundas claimed in claims 14 and 16; and b) formulating this compoundtogether with suitable auxiliaries to give herbicidally orgrowth-regulatory crop protection products.
 20. A method for controllingundesired vegetation and/or for regulating the growth of plants, whichcomprises allowing at least one compound as claimed in claim 17 or 18 ora compound obtainable by the method stated in claim 19 to act on plants,their environment and/or on seeds.
 21. The use of a compound as claimedin claim 17 or 18 or of an agrochemical formulation obtainable by themethod stated in claim 19 in a method as claimed in claim 20 forcontrolling undesired vegetation and/or for regulating the growth ofplants.
 22. A method for generating nucleic acid sequences which i)encode a polypeptide with the biological activity of an NADH-dependentcytochrome b5 reductase which is not inhibited by substances as claimedin claim 17; and which are comprised by a functional equivalent of thenucleic acid sequence of the nucleic acid sequence SEQ ID NO:1 with atleast 52% identity with SEQ ID NO:1; which comprises the followingprocess steps: a) expression, in a heterologous system or in a cell-freesystem, of the proteins encoded by the nucleic acid according to i); b)randomized or site-directed mutagenesis of the protein by modificationof the nucleic acid; c) measuring the interaction of the modified geneproduct with the herbicide; d) identification of derivatives of theprotein which show less interaction; e) assaying the biological activityof the protein after application of the herbicide; and f) selection ofthe nucleic acid sequences which show a modified biological activitytoward the herbicide.
 23. A method as claimed in claim 22, wherein thesequences selected in accordance with claim 22 f) are introduced into anonhuman organism.
 24. A method for generating transgenic plants whichare resistant to substances as claimed in claim 17, which comprisesoverexpressing, in these plants, a nucleic acid sequence encoding apolypeptide with the biological activity of an NADH-dependent cytochromeb5 reductase which comprises a) a nucleic acid sequence with the nucleicacid sequence shown in SEQ ID NO:1; or b) a nucleic acid sequence which,on the basis of the degeneracy of the genetic code, can be deduced fromthe amino acid sequence shown in SEQ ID NO:2 by backtranslation; or c) anucleic acid sequence which, on the basis of the degeneracy of thegenetic code, can be deduced from the amino acid sequence of afunctional equivalent of SEQ ID NO:2 which has at least 39% identitywith SEQ ID NO:2; or d) a functional equivalent of the nucleic acidsequence SEQ ID NO:1 with at least 52% identity with SEQ ID NO:1.
 25. Atransgenic plant generated by a method as claimed in claim
 24. 26. Theuse of compounds having an inhibitory or blocking effect on the activityof an NADH-dependent cytochrome 65 reductase to control undesired plantgrowth and/or regulate the growth of plants.